Pectin extraction from coffee pulp

ABSTRACT

The invention provides a coffee pulp treatment process comprising (a) Providing coffee pulp, obtainable from a production process for producing green coffee beans from coffee cherries; (b) extracting from the coffee pulp a pectin comprising extract, wherein extraction is performed under acid conditions or alkaline conditions, to provide the pectin comprising extract; (c) enzymatic treatment of the pectin comprising extract, wherein the enzymatic treatment comprises a treatment with one or more enzymes selected from the group consisting of an esterase and a reductase, to provide a enzymatically treated pectin material; and (d) extraction of polyphenol functionalized coffee pectin extract from the enzymatically treated pectin material.

FIELD OF THE INVENTION

The invention relates to a coffee pulp treatment process as well as tothe product(s) obtained with such process.

BACKGROUND OF THE INVENTION

With the production of coffee, coffee pulp is produced. Regardless ofthe source (wet or dry processing), by-products and waste products areoften problematic. For example, pulp and mucilage are relatively acidic,corrosive to equipment, and difficult to safely dispose. Furthermore,where the pulp is discarded in a landfill or other disposal site,rotting pulp may lead to unpleasant smells. Therefore, by-products andwaste products have often been viewed as materials which are eitherunusable, hazardous, or of negligible value.

WO2004098320 describes a method for isolating a nutrient from coffeecherries or for producing a food product that comprises a coffee cherryor portion thereof. In WO2004098320, it is particularly preferred thatcoffee cherries will have an extremely low concentration of mycotoxins,including various aflatoxins, fumonisins, ochratoxins, and/or vomitoxin(DON, deoxynivalenol).

SUMMARY OF THE INVENTION

As will be clear from the above, and as also further indicated below,there is a desire to make the coffee production process greener,especially by an economic reuse of parts of the by-products of theprocess, such as coffee pulp. In the process to obtain green coffeebeans, a major stream of biomass is produced. This biomass is rich inuseful bio compounds; however, a technology really commercially readyfor the recovery and use of these compounds is not available. Moreover,the high amounts of toxic compounds (mostly polyphenols and caffeine) inthe streams make the biomass an environmental problem in the coffeeproducing regions. First some general comments are given below.

After collection of the coffee cherries, the coffee cherries aresubjected to various processes to obtain the green coffee bean (i.e.non-roasted coffee bean). Most of the world coffee production isprocessed in two ways; the dry method and the wet method.

In the wet method, the cherries are collected and pulped fresh, leavingthe mucilage (endocarp) and the silver skin attach to the beans; afterpulping the beans go to fermentation tanks for period in general in therange of 12-24 h in which the mucilage is released from the beans andsilver skin. The beans then are dried (sun or hot air dried), the silverskin is removed and the green beans are packed and stored for furthertrade. During these processes about 45% of the coffee cherry biomass isdiscarded as waste material. This biomass has high content ofpolyphenols and caffeine, and therefore becomes toxic in highconcentrations. Although composting is an alternative, big extensions ofland and hard labor are necessary. On top of these requirements, thehigh concentration of polyphenols makes of the use of this compost apoor fertilizer with the risk of poisoning the soil and making itacidic.

Hence, the term “coffee pulp” herein especially refers to the pulpobtained after cherry processing. Therefore, the term “coffee pulp”might also refer to “coffee cherry pulp”. Further, the term “coffeepulp” may also include discarded unripe and overripe cherries, notusable in the production of (high) quality green beans. Hence, the termcoffee pulp may especially refer to one or more of pulp obtained aftercherry processing, discarded unripe cherries not usable in theproduction of (high) quality green beans and discarded overripe cherriesnot usable in the production of (high) quality green beans. The coffeepulp may relate to one or more of the exocarp, outer mesocarp (the pulpitself), and the mesocarp (mucilage or parchment). The hull (also knownas silver skin or parchment) is not taken into account. Note that thesilver skin is part of the seed, not part of the pericarp. The pericarpis especially composed of the skin (exocarp), pulp (outer mesocarp),mucilage (endocarp). The silver skin is part of the endosperm. It isfurther referred to amongst others Esquivel et al, “Functionalproperties of coffee and coffee by-products”, Food ResearchInternational 46 (2012) 488-495, which is incorporated herein byreference. Especially, the coffee pulp is a by-product from the wetmethod processing, or a semi-dry processing, of coffee beans.

In the dry method, the coffee cherries are dried, especially under thesun, for—in general—several days. Thereafter, the dried pulp isseparated from the green beans by pulping. This method does not easilyallow control of the drying process and may therefore generate a low(er)quality coffee.

Most of the coffee nowadays is processed using the wet method (seeabove) with variations to lower water consumption and control over thedrying process (semi-dry, natural pulping etc.). Nevertheless, relativelarge amounts of biomass are still dumped into rivers.

Nowadays, most of the coffee pulp goes without treatment directly tohuge waste disposal sites without any treatment, eventually toxiccompounds from the fermenting cherries leach to the rivers, pollutingthe sources of water in the coffee producing regions. Coffee pulptherefore poses a serious environmental problem, and is a challenge tothe sustainability of the coffee supply chain. With the actualproduction of coffee reaching 10 million tons per year, technology toexploit this waste stream is necessary. Currently coffee pulp is ingeneral used only as compost. There has been research in the use ofcoffee pulp as feed for dairy cattle in Central and South Americas.Nevertheless, these practices use only a small percentage of the wholestream due to the anti-nutritional and toxic compounds in the biomass.Further, it has been suggested in the art to use crude fibers fromcoffee pulp as well as other sub products from this biomass. However,there is no known technology in the extraction separation andmodification of pectin from coffee pulp and mucilage.

Coffee pulp represents 45% of the total weight of the coffee cherry. Thepulp biomass is rich in carbohydrates, polyphenols and caffeine. Becausethe high contents of organic acids, cathechins, and tannins, the coffeepulp and process water pose a serious environmental problem in theregions where production takes place. Coffee discarded streams (the pulpand process water used to separate the mucilage from the bean in the wetmilling factories) have a high BOD (biochemical oxygen demand), whichthreatens water sources. One of the components of coffee pulp is pectin.Pectin is amongst others known in the food industry as gelling agent.However, pectin from coffee pulp has been reported as a poor gellingagent and therefore not useful in food and pharmaceutical applications.It is theorized that the poor gelling properties are a result of theshort length of the pectin backbone, the low molecular weight of thepectin and the high degree of acetylation of the native pectin in thepulp and mucilage of coffee cherry.

Hence, it is an aspect of the invention to provide an alternative coffeepulp treatment process, which preferably further at least partly obviateone or more of above-described drawbacks. It is further an aspect of theinvention to provide an alternative pectin, derived from coffee pulp,that can be used in food applications as gelling agent and/or that mayhave other useful applications. It is further an aspect of the inventionto provide a solution to the coffee pulp, by which the coffee productionprocess can become environmentally more sustainable.

The present invention includes the extraction and use of at least onecompound extracted from the pulp and mucilage after depulping andwashing of the bean, in the wet or semi dry process of green coffeeproduction. Advantageously, the extraction of this bio compound willreduce concentration of toxic compounds in the processing water ofcoffee de-pulping facilities. Further, the extracted compounds in whichpectin is the main component, is a high value ingredient for the foodand/or pharmaceutical industries. Further, it surprisingly appears thatthe extracted bio compounds show the possibility to be tailored forspecific purposes due to the diversity of polysaccharides contained inthe extracted pectin fraction. The pectin obtained with the process ofthe invention may allow applications like as prebiotic as well asgelling agents, but also as mesh for surgical implants are among thepossible uses of the compounds extracted according to the invention.Further, the pectin obtained may be used as thickener or emulsifier.

The technology suggested here aims for the extraction of pectin fromcoffee pulp, and optionally modification (i.e. functionalization) of the(extracted) pectin with enzymes. Such modification may includedemethylation and/or crosslinking the pectins through the esterifiedgroups. The technology presented here especially aims for the extractionof pectin from coffee pulp, and modification of the same pectin withenzymes, to crosslink the pectins through the esterified groups. Theenzymatic modifications surprisingly appear to improve the hydrocolloidal properties of the extracted pectin. The properties of theresulting pectin are very attractive. In the process, the remainingwaste stream may amongst others be detoxified through the hydrolizationof tannins, polymerization of phenols and removal of caffeine during theprocess; this will leave the streams with a substantially reduced BOD(biological oxygen demand) and COD (chemical oxygen demand). Therefore,the environmental impact of coffee production will diminish. Theapproach may also generate income from the exploitation of the biomasswaste as a by-product of the coffee chain. The current invention maysignificantly contribute to improve the sustainability of a major globalcommodity. Hence, the invention provides a biorefinery approach in whichgreen chemistry and biotechnology is applied. The process steps mayconsist of preservation of coffee pulp at the country of production,shipment to a processing site, separation and purification of theproducts, and commercialization of these products in their perspectivemarkets. A market may be the market of food ingredients, wherein highquality pectin as a potential replacement of Arabic Gum is suggested.With the present innovative technology the coffee pectin can be tailoredto meet the standards of different types of applications in the food andpharmaceutical industry. The caffeine content in the remaining waste mayadvantageously be below 10 ppm, such as even below 1 ppm. Hence, theremoval of caffeine may be very efficient while on the other hand also auseful pectin product is provided.

It is known that polyphenols in high concentration can be toxic tocattle, inhibit fermentation and growth of microorganism.Advantageously, in the disclosed invention the presence of polyphenolsis actually desired to allow the modification of the pectin withoutdestroying the biopolymer. The technology is the best alternative at themoment, for the management and exploitation of coffee waste. Therefore,the disclosed technology might be adopted at a big scale. Hence, in afirst aspect, the invention provides a coffee pulp treatment processcomprising:

-   a. Providing coffee pulp, obtainable from a production process for    producing green (i.e. non-roasted) coffee beans from coffee    cherries;-   b. Extracting from the coffee pulp a pectin comprising extract,    wherein extraction is performed under acid conditions or alkaline    conditions (or one after the other), to provide (or produce) the    pectin comprising extract, especially wherein the extraction    comprises extracting from the coffee pulp a pectin comprising    extract, wherein extraction is performed under (at least) acid    conditions;-   c. (optionally) enzymatic treatment of the pectin comprising    extract, wherein the (optional) enzymatic treatment comprises a    treatment with one or more enzymes selected from the group    consisting of an esterase and/or a reductase, to provide an    enzymatically treated (or modified) pectin material, especially    polyphenol functionalized coffee pectin extract, especially wherein    the enzymatic treatment comprises at least a treatment with an    oxidoreductase; and-   d. (optionally) extraction of polyphenol functionalized coffee    pectin extract from the product of the (optionally) enzymatic    treated pectin comprising extract (i.e. the product obtained at c).

With this process, advantageously in an embodiment polyphenolfunctionalized coffee pectin extract is produced, which is a productthat can be used for several applications, and which leads to aremaining product that has a substantially reduced content inpolyphenols, and may therefore be more easily reused or discarded aswaste.

The coffee pulp that is used for the process may directly be obtainedfrom a plant, but may also have been subjected to a conservationprocess. The coffee pulp used may also be obtained from a remote place(like >10 km, or even >100 km or even further), and after transportationbe used as coffee pulp in the process of the invention. Beforetransportation, the coffee pulp may optionally be treated forconservational purposes.

The extraction per se, especially including an alkali and acidicprocedure, see also below, is also an aspect of the invention.Especially, the enzymatic treatment is applied, which may be used todemethylate and/or cross-link.

The term “green coffee bean” is known in the art and especially refersto the non-roasted coffee bean. The cherries that are used in de-pulpingmay be in a ripe or unripe state. Also mixtures of unripe and ripecherries may be applied. The properties of the polyphenol functionalizedcoffee pectin extract may depend upon whether ripe and/or unripe coffeebeans are applied.

Pectin can be extracted from multiple sources, however pectins aremostly extracted from citrus peels and apple pomace. As mentioned above,pectins are chemically and/or enzymaticaly modified to obtain desiredgel structures. Another source of pectin that has been accepted ispectin extracted from the industrial residues of sugar from beetroot(SBP). Physicochemical differences between SBP and other type ofconventional pectins include higher proportion of neutral sugar sidechains, a higher content of acetyl groups at O2 and O3 positions withinthe galacturonic backbone and a higher content of phenolic esters in theside chains particularly in the arabinose and galactose, and a highercontent of protinaceous materials bound to the side chains throughcovalent linkages. Unexpectedly, coffee pectin shares some of thecharacteristics intrinsic to SBP, the low molecular weight of the pecticmolecules, the presence of important amounts of galactose and arabinosein the neutral side chain, and the presence of polyphenols among others.It is therefore theorized that coffee pectin can be modified as SBP andyield high value pectins with emulsifying characteristics. Also coffeepectin can be chemically modified as citrus peel pectin to produce thedesired gel, in this aspect research and standardisation are stillneeded.

Instead of coffee pulp, also other pectin comprising agriculturalby-products may be applied, especially pectin extracted from theindustrial residues of cacao, palm oil, olive oil and sugar frombeetroot (SBP). Hence, in another aspect, the invention provides apectin comprising agricultural by-product treatment process comprising:

-   a. Providing a pectin comprising agricultural by-product;-   b. Extracting from the pectin comprising agricultural by-product a    pectin comprising extract, wherein extraction is performed under    acid conditions or alkaline conditions (or one after the other), to    provide the pectin comprising extract, especially wherein the    extraction comprises extracting from the coffee pulp a pectin    comprising extract, wherein extraction is performed under (at least)    acid conditions;-   c. (optionally) Enzymatic treatment of the pectin comprising    extract, wherein the enzymatic treatment comprises a treatment with    one or more enzymes selected from the group consisting of an    esterase and a reductase, to provide a enzymatically treated pectin    material, especially wherein the enzymatic treatment comprises at    least a treatment with an oxidoreductase; and-   d. (optionally) Extraction of polyphenol functionalized coffee    pectin extract from the product of the optionally enzymatic treated    pectin comprising extract.

The invention will further be elucidated with respect to (pectincomprising) coffee pulp.

In a specific embodiment, the coffee pulp is subjected to a firstextraction under acid conditions, leading to a first extraction productand a residual product, wherein the residual product is furthersubjected to a second extraction under alkaline conditions, leading to asecond extraction product and a second residual product, and wherein thefrom this second extraction obtained second extraction product isoptionally recombined with the first extraction product from the firstextraction, and wherein these optionally combined pectin extractionproducts are then further subjected to the enzymatic treatment.Especially, in the second extraction a second extraction liquid isapplied that comprises H₂O₂. H₂O₂ may be used as oxidizing agent and/oras substrate for the enzyme(s). H₂O₂ may allow e.g. the reaction of alaccase and/or a peroxidase, especially a peroxidase, for cross linkingHowever, other oxidizing agent can be used (oxygen donors).Alternatively or additionally to H₂O₂ also ammonium persulfate((NH₄)₂S₂O₈) and/or sodium metabisulfite (Na₂S₂O₅) may be applied.Optionally or additionally, also ozone might be applied. The extractionmay also include a separation step separating the extract from theremaining product, such as by filtration etc. (see also below).

The term “acidic conditions” and similar terms especially indicate apH<7; the term “alkaline” conditions and similar terms especiallyindicate a pH>7.

As can be derived from the above, in an embodiment the method mayinclude extracting (from the pectin comprising agricultural by-product)a pectin comprising extract, wherein extraction is performed under acidconditions to provide the pectin comprising extract, followed by theenzymatic treatment. The additional extraction under alkaline conditionsis a specific embodiment. As can be derived from the above, also in anembodiment the method may include extracting (from the pectin comprisingagricultural by-product) a pectin comprising extract, wherein extractionis performed under alkaline conditions, to provide the pectin comprisingextract, followed by the enzymatic treatment. The additional extractionunder acid conditions is a specific embodiment. The acid extractionprocess especially provides a pectin that is useful for the foodindustry. The (additional) (dilute) alkali extraction may assist inextracting pectin that has low solubility in water.

As can be derived from the above, in an embodiment the method mayinclude extracting (from the pectin comprising agricultural by-product)a pectin comprising extract, wherein extraction is performed under acidconditions and alkaline conditions, to provide the pectin comprisingextract which is a combination of the alkaline extraction product andacid extraction product, followed by the enzymatic treatment (of thecombination of extracts). As will be discussed below, the acidextraction may be subsequent to the alkaline extraction or the alkalineextraction may be subsequent to the acid extraction. The phrase “whereinextraction is performed under acid conditions and alkaline conditions”in general indicates that first an extraction is performed under acid oralkaline conditions and that (subsequently) the remaining material fromthe acid or alkaline extraction is subjected to an alkaline or acidextraction, respectively. The extracts can be combined for further(enzymatic) processing and the remaining material can be used for otherapplications or discarded (see elsewhere herein).

In a preferred embodiment, the acid conditions of the first extractionare at a pH in the range of 0.5-4, especially 1.5-3. Further, the firstextraction may especially be performed at a temperature of at least 80°C. The alkaline conditions in the second extraction are especially at apH in the range of 7-14, such as 8-14, even more especially 7-11, suchas 9-11, such as especially between 7.5-10.5, such as 9.5-10.5. For thealkaline extraction, the pH is >7, especially 7.5-9. At larger pH, thepectin molecule may start getting hydrolyzed. Further, especially thealkaline extraction is performed at a temperature not higher than 45° C.especially 35° C.

Further, in embodiments the one or more enzymes are selected from thegroup consisting of diphenol oxidoreductase, peroxidase, laccase,pectin-esterase, methyl-esterase, poly galacturanase, endopolyglucanase, and exo polyglucanase. Alternatively or additionally, oneor more enzymes selected from the group consisting of pectin lyase,polygalacturonase (endo and exo), endo galactanase, exo galactanase,rhamnogalacturonase may be applied. Alternatively or additionally,especially arabinofuranosidase, arabinase, feruloyl esterase, endopectin methyl esterase, exo pectin methyl esterase, pectin esterase,laccase, peroxidase (especially from horseradish) may be applied. Fordemethylation, especially enzymes like pectin methyl esterase (EC3.1.1.11) can be applied. For cross-linking, especially enzymes likeperoxidase (especially EC 1.10 or EC 1.11, such as e.g. horseradishperoxidase 1.11.1.7) may be applied. Especially, the enzymatic treatmentat least involves a treatment of the extract(s) with an oxidoreductase.An oxidoreductase is an enzyme that catalyzes the transfer of electronsfrom one molecule (reductant or electron donor) to another the molecule(oxidant or electron acceptor). Best results are obtained with anoxidoreductase selected from the (sub)classes EC 1.10 (oxidoreductasesthat act on diphenols and related substances as donors) and EC 1.11(oxidoreductases that act on peroxide as an acceptor (peroxidases)).Especially good results were obtained with horseradish peroxidase(1.11.1.7) and laccase (EC 1.10.3.2). The former may need H₂O₂ whereasthe latter may use dissolved oxygen and may not necessarily need anadditional oxygen donor. Hence, especially the enzymatic treatmentcomprises at least a treatment (of the pectin comprising extract) withone or more enzymes selected from the group consisting of EC 1.10 or EC1.11. The EC 1.10 class enzymes (or enzymatic reactions) are acting ondiphenols and related substances as donors, with e.g. NAD+, NADP+,cytochrome, oxygen, copper or other acceptors. Especially those withoxygen as acceptor are used. The EC 1.11 class enzymes (or enzymaticreactions) are acting on peroxide as acceptors. Here, as will be knownto the person skilled in the art, the international accepted enzymenomenclature, such as especially defined by the Nomenclature Committeeof the International Union of Biochemistry and Molecular Biology(NC-IUBMB), is applied.

However, the process may also be performed in an alternative way,wherein the alkaline and acid extraction order is reversed. Hence, inyet another embodiment, the coffee pulp is subjected to a firstextraction under alkaline conditions, leading to a first extractionproduct and a residual product, wherein the residual product is furthersubjected to a second extraction under acid conditions, leading to asecond extraction product and a second residual product, and wherein thefrom this second extraction obtained second extraction product isoptionally recombined with the first extraction product from the firstextraction, and wherein these optionally combined pectin extractionproducts are then further subjected to the enzymatic treatment.Especially, during the first extraction a first extraction liquid isapplied that comprises H₂O₂. As indicated above, H₂O₂ may be used asoxygen donor and/or as substrate for the enzyme(s). In a preferredembodiment, the alkaline conditions on the first extraction areespecially at a pH in the range of 7-14, such as 8-14, especially 7-11,such as 9-11, more especially 7.5-10.5, such as 9.5-10.5. For thealkaline extraction, the pH is >7, especially 7.5-9. At larger pH, thepectin molecule may start getting hydrolyzed. Further, especially thealkaline extraction is performed at a temperature not higher than 45° C.especially 35° C. Further, especially the acid conditions of the secondextraction are at a pH in the range of 0.5-4, especially 1.5-3. Infurther embodiments, the second extraction is especially performed at atemperature of at least 80° C.

The enzyme may be used in one or more of the following instances: duringthe acid extraction, after the acid extraction, during the alkalineextraction, after the alkaline extraction, and during a stage when bothextracts have been combined. Of course, during one or more of thesestages, an enzyme may be applied. Especially, the enzyme, and optionaladditive for the enzyme such as H₂O₂, may depend upon the pH and/ortemperature. H₂O₂ may for instance only be applied when peroxidase isused, especially horse radish peroxidase. Laccase does not need H₂O₂ togenerate the polyphenol cross-links. However, laccase in general onlysubstantially acts at pH between about 6.0 to 8.5. Horse radishperoxidase acts in general only substantially at pH higher between about8.5 and 12.5. The extraction pH may thus e.g. also depends in the enzymeused, though, if necessary, after extraction the pH may also be alteredto arrive at a pH suitable for the chosen enzymes. Therefore, inembodiments wherein (horse radish) peroxidase is applied, the presenceof H₂O₂ is desired and the pH range, during at least part of theprocess, is especially from 6.0 to 11 since this the range where thereductase is more active. The temperature in the alkali extraction isespecially not over 45° C. Further, the optimum temperature for bothlaccase and peroxidase is in the range of 30-40° C., such as about 35°C. Enzymes may be added during any stage of the process, but are ofcourse at least available during the enzymatic treatment.

Yet in further embodiments, the one or more enzymes are especiallyselected from the group consisting of diphenol oxidoreductase,peroxidase, laccase, pectin-esterase, methyl-esterase, polygalacturanase, endo polyglucanase, and exo polyglucanase. Alternativelyor additionally, one or more enzymes are selected from the groupconsisting of pectin lyase, polygalacturonase (endo and exo), endogalactanase, exo galactanase, rhamnogalacturonase may be applied.Alternatively or additionally, especially arabinofuranosidase,arabinase, feruloyl esterase, endo pectin methyl esterase, exo pectinmethyl esterase, pectin esterase, laccase, peroxidase (especially fromhorseradish) may be applied. For demethylation, especially enzymes likepectinesterase can be applied. For cross-linking, especially enzymeslike peroxidases may be applied. As indicated above, during theenzymatic treatment especially at least an oxidoreductase selected fromthe (sub)classes EC 1.10 and EC 1.11 is applied. Note that the term “anenzyme” or “an oxidoreductases” and similar terms may also refer to aplurality of (different) enzymes or a plurality of (different)oxidoreductases, etc., respectively. As indicated herein, the enzymatictreatment may for instance be during an extraction stage or subsequentto an extraction stage, or multiple enzymatic treatments may be applied.Further, also a cocktail of different enzymes may be added. Assuming aoxidoreductases, the amount of enzyme used will be in the range of about0.5-10 mg, such as especially about 1 mg of pure enzyme (100% protein)per 100 ml and assuming an esterase, the amount of enzyme used will bein the range of about 1-10 mg, especially about 5 mg of pure enzyme(100% protein) per 100 ml Hence, the enzymatic modification may beexecuted in a reactor, where there is an actual transformation of thematter. In one or more of the alkali and acid extraction there may be no(enzymatic) modification. Therefore, these may be executed in anextraction unit. During the enzymatic modification step the enzyme(s)may especially transform the pectin to a cross-linked pectin and/or maycleave groups attached to the pectin (macromolecules).

In a further specific embodiment, prior to the (first) extraction, thecoffee pulp is subjected to a washing process, wherein the washingprocess comprises mixing the coffee pulp with a solvent and subsequentlyremoving at least part of the solvent, wherein the water content of thesolvent is ≦80 wt. %. Especially, at least 50 wt. %, even moreespecially at least 80 wt. %, of the solvent consists of one or moreliquids having a polarity lower than water (vide infra)

In an embodiment, the extraction of polyphenol functionalized coffeepectin extract from the product of the optionally enzymatic treatedpectin comprising extract comprises mixing at least part of theenzymatically treated material with an extraction liquid andsubsequently removing at least part of the polyphenol functionalizedcoffee pectin, wherein the extraction liquid has a pH in the range of3.5-6, such as 4-6. Especially, the extraction liquid comprises ethanol.However, the extraction liquid may also comprise other solvents, such asmethanol, 2-propanol, acetone. The same type of solvent may be used asused for the washing process (see also below). Further, the extractionliquid may be acidified. Further, the extraction liquid may comprise acombination of two or more of (such) solvents. This extraction liquidmay be used to precipitate the functionalized pectin. By adding thesolvent, pectin may precipitate creating a gel which can be separatedfrom the low molecular weight compounds dissolved in the solvent.

With the process of the invention, but optionally via other routes, apolyphenol functionalized coffee pectin extract may be obtained. Hence,in a further aspect, the invention also provides a polyphenolfunctionalized pectin (per se), especially a polyphenol functionalizedcoffee pectin. Hence, especially the invention provides a polyphenolfunctionalized coffee pectin obtainable by the process as describedherein. Especially, the polyphenol functionalized coffee pectin has amolar ratio of phenolic groups to the sum of arabinose plus galactoseunits between 20% to 60%, and has a molecular weight >90,000 Da. In thepectin (product) of the invention, the protein content may be in therange of 5-18 wt. %, especially 8-15 wt. %. Further, the polyphenolcontent may be in the range of 0.06-0.18 wt. %, especially 0.09-0.15 wt.%. As known in the art, the protein content can be determined based onthe Dumas method (ISO 16634-1:2008); the polyphenol content can bedetermined based on the Folin-Ciocalteu (ISO 14502-1:2005) method withthe Folin-Ciocalteu reagent (FCR) or Folin's phenol reagent orFolin-Denis reagent, also called the Gallic Acid Equivalence method(GAE). This reagent (method) is especially designed for determining thephenol amount. Especially, the invention further provides a polyphenolfunctionalized coffee pectin (as described herein), having a molarweight <200,000 Da, such as especially <120,000 Da, such as in the rangeof 90,000-120,000 Da. The coffee pectin may have a total sugar contentof rhamnose, arabinose, xylose, mannose, galactose, glucose,galacturonic acid, relative to the total sugar content, in the range of70-95 wt. %, with a total galacturonic acid content, relative to thetotal sugar content, in the range of 55 to 80 wt. %. Further, the totalglucose content, relative to the total sugar content, may be in therange of 3-15 wt. %. The total sugar content in the pectin (product) ofthe invention may be in the range of 40-80 wt. %, relative to the totalweight of the product. The remaining part may include amongst otherspolyphenol and protein. Further, especially the Gal/UA ratio may be inthe range of 0.1-0.2 (galactose-uronic acid weight ratio).

In addition to the above, further (intermediate) process steps may beincluded, such as one or more of precipitation, filtration, washing andresuspension. For instance, after recombination of the two extracts (orafter acid extraction, but) before an enzymatic treatment, (also) aprecipitation, filtration, washing and resuspension may take place. Theterms “upstream” and “downstream” relate here to an arrangement of itemsor features, or a sequence of stages, relative to the propagation of aprocess chain, wherein relative to a first stage within a chain ofprocess actions or process apparatus or process stages, a second stagein the process chain closer to the beginning of the chain is “upstream”,and a third stage within the process chain further away from the processbeginning is “downstream”.

The term “substantially” herein, such as in “substantially all emission”or in “substantially consists”, will be understood by the person skilledin the art. The term “substantially” may also include embodiments with“entirely”, “completely”, “all”, etc. Hence, in embodiments theadjective substantially may also be removed. Where applicable, the term“substantially” may also relate to 90% or higher, such as 95% or higher,especially 99% or higher, even more especially 99.5% or higher,including 100%. The term “comprise” includes also embodiments whereinthe term “comprises” means “consists of”. The term “and/or” especiallyrelates to one or more of the items mentioned before and after “and/or”.For instance, a phrase “item 1 and/or item 2” and similar phrases mayrelate to one or more of item 1 and item 2. The term “comprising” may inan embodiment refer to “consisting of” but may in another embodimentalso refer to “containing at least the defined species and optionallyone or more other species”.

Furthermore, the terms first, second, third and the like in thedescription and in the claims, are used for distinguishing betweensimilar elements and not necessarily for describing a sequential orchronological order. It is to be understood that the terms so used areinterchangeable under appropriate circumstances and that the embodimentsof the invention described herein are capable of operation in othersequences than described or illustrated herein.

The apparatus herein are amongst others described during operation. Aswill be clear to the person skilled in the art, the invention is notlimited to methods of operation or devices in operation.

It should be noted that the above-mentioned embodiments illustraterather than limit the invention, and that those skilled in the art willbe able to design many alternative embodiments without departing fromthe scope of the appended claims. In the claims, any reference signsplaced between parentheses shall not be construed as limiting the claim.Use of the verb “to comprise” and its conjugations does not exclude thepresence of elements or steps other than those stated in a claim. Thearticle “a” or “an” preceding an element does not exclude the presenceof a plurality of such elements. The invention may be implemented bymeans of hardware comprising several distinct elements, and by means ofa suitably programmed computer. In the device claim enumerating severalmeans, several of these means may be embodied by one and the same itemof hardware. The mere fact that certain measures are recited in mutuallydifferent dependent claims does not indicate that a combination of thesemeasures cannot be used to advantage.

The invention further applies to an apparatus comprising one or more ofthe characterizing features described in the description and/or shown inthe attached drawings. The invention further pertains to a method orprocess comprising one or more of the characterising features describedin the description and/or shown in the attached drawings.

The various aspects discussed in this patent can be combined in order toprovide additional advantages. Furthermore, some of the features canform the basis for one or more divisional applications.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will now be described, by way of exampleonly, with reference to the accompanying schematic drawings in whichcorresponding reference symbols indicate corresponding parts, and inwhich:

FIG. 1 schematically depicts an embodiment of the process as describedherein;

FIGS. 2 a-2 b schematically depicts coffee pectin (before and afterprocessing as described in some embodiments herein);

FIG. 3 shows a high performance size exclusion chromatography (HPSEC)for commercial pectin from citrus peel and pectin obtain with the hereindescribed process;

FIG. 4 shows the presence of oligomers after digestion of pectin withpolygalacturonase from Aspergilius aculetus after high performance anionexchange chromatography (HPAEC) for commercial pectin from citrus peeland pectin obtain with the herein described process;

FIG. 5 a schematically shows a coffee bean;

FIG. 5 b shows a (Laboratory) scale process for fresh material;

FIG. 5 c shows the oxidation of cathecol by PPO from coffee pulp in time(in absorbance units, y-axis, and with time in hours on the x-axis).Data are obtained spectrophotometrically at 420 nm. The sing ⋄ indicatesthe absorbance change of the enzyme extract (indicated with square),minus the absorbance change of the substrate solution (indicated withtriangle), without enzyme;

FIG. 5 d shows a flow chart for large-scale preservation of dried coffeepulp; and

FIG. 5 e shows a large scale extraction from preserved wet pulp (withmass balance). The schematic drawings herein are not necessarily onscale.

DETAILED DESCRIPTION OF THE EMBODIMENTS

FIG. 1 schematically depicts an embodiment of the process as describedherein. Blended coffee pulp: the coffee pulp may relate to one or moreof the exocarp, outer mesocarp (the pulp itself), and the mesocarp(mucilage or parchment). The hull (also known as silver skin) is nottaken into account (later during the process, hulling machines may beused to remove the parchment layer) from wet processed coffee. Pulpingdry processed coffee refers to removing the entire dried husk—theexocarp, mesocarp & endocarp—of the dried cherries. Further, optionallypolishing may take place; this is an optional process in which anysilver skin that remains on the beans after pulping (optionally apolishing machine may be applied). The pulp is preferably collected fromthe depulping mills as soon as possible after generation of the pulp,preferably in the first 24 hours. Would this not be possible, it ispreferred to use preservation steps to minimize pectin degradation dueto enzymatic activity. The preservation steps can be but are not limitedto: acidification of the pulp to a pH below 4, alkalinisation to a pHabove 9, lowering of the water activity to aw=0.5 or less attemperatures below 60° C., inactivation of the endogenous enzymes bysolvent addition, inactivation of the endogenous enzymes by boilingand/or cooling (cycles). Addition of one or more preservation agentsselected from the group consisting of sodium meta bisulfite, ascorbicacid, ethylenediaminetetraacetic acid (EDTA) may also be applied.Examples of (further preservation agents are e.g. selected from thegroup consisting of ascorbic acid, citric acid, oxalic acid, sodiummetabisulfite, potassium bisulfite, sulfur dioxide, glycine, methionineand EDTA, especially one or more of sodium metabisulfite, potassiumbisulfite, and EDTA.

Hence, in embodiments of the process, prior to the extraction (butespecially after washing) the pulp is subjected to a preservationprocess, wherein the preservation process comprises one or more of (i)adding a preservation agent to the pulp and (ii) drying the pulp. Asindicated above, the preservation agent may comprise one or more ofascorbic acid, citric acid, oxalic acid, sodium metabisulfite, potassiumbisulfite, sulfur dioxide, glycine, methionine and EDTA, especially oneor more of sodium metabisulfite, potassium bisulfite, and EDTA,especially one or more of sodium metabisulfite and potassium bisulfite.Further, it is preferred that the pulp is milled (or macerated) to asuitable particle size (after preservation but) prior to any processingstep or stage. A suitable particle size is in the range between 10 and40, such as e.g. 18 mesh. The coffee pulp is indicated with reference10. The use of milled pulp may lead to a better extraction than whenusing unmilled pulp.

The (optional) clean-up procedure, herein also indicated as washingprocess, indicated with reference 100, may comprise the treatment of the(blended) coffee pulp with a solvent of low polarity, it can be, but isnot limited to one or more of acetic acid, acetone, acetonitrile, acetylacetone, 2-aminoethanol, aniline, anisole, benzene, benzonitrile, benzylalcohol, 1-butanol, 2-butanol, i-butanol, 2-butanone, t-butyl alcohol,carbon disulfide, carbon tetrachloride, chlorobenzene, chloroform,cyclohexane, cyclohexanol, cyclohexanone, di-n-butylphthalate,1,1-dichloroethane, diethylene glycol,1-Methoxy-2-(2-methoxyethoxy)-ethane (diglyme), dimethoxyethane (glyme),N,N-dimethyl aniline, dimethyl formamide (DMF), dimethyl phthalate,dimethyl sulfoxide (DMSO), dioxane, ethanol, ether, ethyl acetate, ethylacetoacetate, ethyl benzoate, ethylene glycol, glycerin, heptane,1-heptanol, hexane, 1-hexanol, methanol, methyl acetate, methyl t-butylether (MTBE), methylene chloride, 1-octanol, pentane, 1-pentanol,2-pentanol, 3-pentanol, 2-pentanone, 3-pentanone, 1-propanol,2-propanol, pyridine, tetrahydrofuran (THF), toluene, such as especiallyone or more of methanol, a methanol water mixture, hexane, toluene,ethylene glycol, ether, ethyl ether. The solvent may optionally beacidified. The pulp is mixed with the solvent, preferably in a countercurrent extractor. The hydrocinnamic acids as well as the freepolyphenols are dissolved. In this stream there may also be a richfraction of caffeine which in later stage can be purified, and might bea sub product of the pectin extraction. The product obtained after thewashing process is indicated with reference 110. Washing with a solventmay remove free polyphenols and caffeine from the coffee pulp and it mayalso precipitate polysaccharides of higher degree of polymerization.Washing with solvent may remove as much free polyphenols as possiblewithout removing the polysaccharides that have polyphenols in theirfunctional groups. Bottom line is solvent of lower polarity such asethanol and propanol, precipitate the pectin with polyphenols attachedwhile solubilizing caffeine and polyphenols that are not attached to thepectin structure. This allows to later use enzymes such as laccase toselectively modify pectin with biphenolic groups without loosing toomuch enzyme polymerizing the high amount of polyphenols. The washingliquid for the pulp may especially comprise less than 70 wt. % water,especially less than 65 wt. % water, such as less than 55 wt. %, or evenlower. The liquid(s) used for the extraction(s) especially have a higherwater content than the washing liquid (for the pulp). For instance, theextraction liquid(s) may comprise more than 65 wt. %, especially morethan 75 wt. %, even more especially at least 80 wt. %, such as at least90 wt. %, like at least 95 wt. % water. Further, the liquid(s) used forthe extraction(s) especially have a higher polarity than the washingliquid (for the pulp). In this way, free polyphenols and caffeine may beremoved from the pulp by the washing liquid and by extraction with apolar solvent (especially an acid(ified) polar solvent) pectin may beextracted from the insoluble solids of the pulp. Further, as indicatedabove, the extraction liquid(s) especially has a pH <7 or a pH >7.Optionally, the extraction liquid(s) may also comprise a (solved) salt(see also below).

In a first extraction step or stage, indicated with reference 200, thebiomass, especially the product obtained after the washing process, mayin an embodiment, be acidified to a pH=4 or lower, especially a pH=2 orlower, with a concentrated acid such as but not limited to one or moreof hydrochloric acid, nitric acid, phosphoric acid, acetic acid.Further, the mixture may be heated, such as to a temperature of at least70° C. The pH is preferably lowered before the heating step or stage canbe applied. The ratio of biomass to (extraction) liquid may especiallybe in the range of 0.25:1-1:0.25, especially 0.5:1-1:0.5, such as 1:1(which means that for each kilogram of fresh pulp one liter of solutionis necessary for the extraction). It is preferably to use a highconcentrated buffer solution to mix the biomass and then adjust the pH.Possible salts solutions are (but not limited) to one or more of sodiummono basic phosphate (NaH₂PO₄), sodium nitrate, sodium acetate, andsodium chloride. Alternatively or additionally, ammonium and/orpotassium salts may be applied. Of course, more than one salt may beapplied. The concentration of the salts may range between 50-400 mM,such as especially 100 mM to 200 mM (for each salt individually).Extraction may e.g. be executed in an extraction vessel or a countercurrent extractor where liquids and solids are mixed together and mixedcontinuously. It is desirable that heating is performed as fast aspossible. Therefore, pre heating of the extraction vessel may beadvisable. The product obtained after the first extraction step or stageis indicated with reference 210.

It is preferred that the first separation step or stage, indicated withreference 300, is performed on the hot mix (obtained in the firstextraction step or stage). It is desired to recover as much solution(i.e. filtrate or supernatant) as possible before continuing with thenext step or stage. According to the setup and magnitude of the streamdifferent types of separation units can be used. Examples are a frameseparator, a plate separator, a sieve (separator) and a centrifuge(separator). The solid precipitating from a liquid is called aprecipitate (residual product, or first residual product), or whencompacted by a centrifuge, a pellet. The liquid remaining above thesolid is in either case called the supernate or supernatant. Alsofiltration with a filter may be performed. The process of passing amixture through a filter is called filtration. The liquid produced afterfiltering, in general a suspension of a solid in a liquid, is calledfiltrate, while the solid remaining in the filter is called retentate,residue, or filtrand. The remaining liquid after the first separation,the supernatant or filtrate (here the first extraction product), goes toa reactor in which it may be mixed with the supernatant of a secondseparation step or stage. The precipitate, retentate, residue, sedimentor filtrand (first residual product) must especially be composed of onlysolid matter as much as possible. At this point the (remaining) biomassshould be approximately 50% to 75% of the starting mass (dry weight).

The products obtained after the first separation step or stage areindicated with references 310 and 320. Reference 310 refers to theproduct that is remaining after the first separation, such as aretentate, residue, or filtrand, sediment, etc. This product 310 (firstresidual product) is especially subjected to a second extraction 400,see also below. The (desired) product, indicated with reference 320, ofthe separation action, i.e. a filtrate or permeate, or supernatant, etc.(first extraction product) can be directly introduced in a first reactor600 (or reaction stage), see also below. The pectin comprising (extractafter separation), in this embodiment indicated with reference 320, is aliquid product (extraction liquid with extract)

In a second extraction step or stage, indicated with reference 400, thefirst extraction residual product 310 or biomass may be mixed withalkali to extract the more ramified polysaccharides as well as moreesterified pectins, which comprised the coffee pulp. This may in anembodiment be done in several steps or stages. First with a(nextraction) liquid, especially water, the biomass is diluted until 50%of the total dilution is achieved. Thereafter, the pH may be adapted,e.g. with concentrated alkali (i.e. an alkali solution), to a value ofespecially at least 9, like e.g. 10. In an embodiment, after adding thealkali, hydrogen peroxide may be added up to especially a concentrationof up to 7.5%, especially up to 5% of the starting biomass. In the laststage of this extraction, the volume is completed with water. Extractionmay be executed in an extraction vessel, a extraction vessel may becomposed of a recipient in adequate material, such as stainless steel320, 316 or alloys that prevents rust. A recirculating pump may or maynot be present depending of the operation if continues or batch. Theextraction vessels must include a source of heat and mixing mechanism.Mixing should be promoted to obtain higher rates of delignification andhydrolysis of the esterified compounds attach to the pectins. Thetemperature of the extraction vessel is especially regulated to avoidbreakdown of the biopolymer. A suitable temperature is in the range of35-65° C. The alkali concentration (of the concentrated alkali solution)is especially approximately 6 molar to 8 molar. The alkali (solution)can be based on a solution of e.g. one or more of sodium hydroxide,potassium hydroxide, calcium carbonate, and ammonium acetate. Theproduct (mixture) obtained after the second extraction step or stage isindicated with reference 410.

In a second separation step or stage the aim is especially to separatethe solids (from the product (mixture) obtained after the secondextraction), i.e. the second (extraction) residual product, which aremostly cellulose and lignin from the free polysaccharides that are insolution (second extraction product), if necessary the pH can be loweredto 7 or 8 before separation. A lowering of the pH may be controlled to(substantially) prevent gelation of the pectins, which may lead to aloss of the biopolymers with the retentate. The second separation stepor stage is indicated with reference 500. According to the setup andmagnitude of the stream different types of separation units can be used.Examples are a frame separator, a plate separator, a sieve (separator)and a centrifuge (separator). The (desired) product of the (second)separation action, i.e. a filtrate or permeate, or supernatant, etc.(i.e. the second extraction product), indicated with reference 520, can(also) directly introduced in a first reactor 600. The second(extraction) residual product, not indicated, can be discarded.

Note that in this schematically indicated process the second extractionstage 400 is downstream of the first reaction stage. Note however thatthe acid and alkaline extractions may also be performed in anotherorder, i.e. the first reaction stage including an alkaline extractionand the second reaction stage including an acid extraction. The productobtained after alkaline and acid extraction (or acid an alkalineextraction), may also be relevant per se. However higher quality pectinsmay be obtained when also the enzymatic processing as defined herein isapplied. Alkaline extraction may optionally be omitted, acid extractionhowever is especially desired.

In a first reactor (herein also indicated as reactor 600, bothacid-extracted pectins and if present alkali-extracted pectins may bemixed. Mixing may be done in ways known to the person skilled in theart, like with an extruder or a stirrer. Hence, the first reactor mayespecially include one or more of an extruder and a stirrer. Due to thechange of pH some pectins can gel. Hence, it is especially preferred tolower the pH (of the alkaline liquid) slowly to a point near neutrality,especially in the range of 6-8 pH, preferably between pH 6.5-7.0. Asindicated above, optionally directly after acid extraction, theenzymatic treatment may be executed. In such instance, the pH of theseparation product may be increased to a pH in the range of 6-8preferably between pH 6.5-7.0.

In the first reactor, especially two types of enzymes, oxidoreductaseand/or esterase, are applied separately or in combination.Oxidoreductase is added according to its activity, i.e. the necessaryamount to react with the pectin polymer is added. Examples of theoxidoreductase are (but not limited) to: diphenol oxidoreductase,peroxidase, and laccase. Esterase, especially pectin esterase, is addedto control the degree of methylation and esterification of the pectin.Example of the (esterase) enzymes are (but not limited) to:pectin-esterase, methyl-esterase, poly galacturanase, feruloyl esterase,arabinose, arabinofuranosidase and endo and exo polyglucanase (see alsoabove). As indicated above, at least an oxidoreducatase may be applied,even more especially in combination with an esterase (especiallyPectinesterase (EC 3.1.1.11)). In addition to the enzyme(s) also one ormore further additives may be added. As indicated above, the pH may bechanged (if necessary) to approximately neutral. Also other enzymes thandefined herein, having the same functionality may be applied.

The product obtained after processing in the first reactor, or afterprocessing in this reaction stage, is indicated with reference 610. Thisproduct may be subjected to a next reaction stage, which is indicatedwith reference 700. Herein, references 600 and 700 may (also) refer todifferent reactors, respectively. However, these references may alsorefer to reaction stages, which may in an embodiment be performedconsecutively in different reactors whereas heat source, pH control andthermostat are present or in the same reactor. Reference 700 may alsorefer herein to a second reactor (or vessel).

In a second reactor (indicated with reference 700), a solvent, such asethanol, methanol, 2-propanol, acetone especially an acidified solvent,such as acidified ethanol (like ethanol +1% Acetic acid anhydrous) isadded, especially in the ratio of 1:1-1:10, such as 1.2:1:6, like 1:4extraction solution to (acidified) solvent, such as ethanol. Thefunction of the solvent is especially to change the polarity of thesolution so the pectin will precipitate creating a gel which can beseparated from the low molecular weight compounds dissolved in thesolvent. The mix is left for coagulation of the pectins andprecipitation. It is preferred that the pH in this stage is lower than6; however is not advisable to have a pH lower than 3. If necessary, thesolvent (or extraction liquid) preferably has a concentration of alcoholof 70% or higher. In an embodiment, the (second) reactor is or comprisesalso a decanter. In this way, the upper layer of the solution can bedisposed leading to much smaller volume for the last separation step orstage.

In a third separation step or stage, indicated with reference 800, theunrefined pectin, indicated with reference 710, is separated from thesolvent(s), such as ethanol and other solvents used during the process.Due to the colloidal characteristics of the pectins, in an embodiment acentrifuge may be applied for separating the pectins from thesolvent(s). The product thus obtained (here the retentate, filtrand orsedimentation, etc.) is indicated with reference 810, which comprisesthe polyphenol functionalized (coffee) pectin. The product for thisreaction stage (or this reactor) is indicated with reference 810, andcan be indicated as the third extraction product (which is a solidmaterial). Again, reference 800 may also refer to a further reactor, athird reactor which must be constructed in resistant material such asstainless steel 316, and 320, fire proof and suitable to work withvolatile solvents. However, references 700 and 800 may also refer to areactor including a decanter.

If desired, the pectins thus obtained can further be refined to meetspecification in different industries. For instance, higher molecularweight pectins can be obtained by further crosslinking with (purified)enzymes and/or gelling pectins can be de-esterified to meet differenttypes of application in the food and beverage industries. Furthermore,pectin can be modified with arabinase and arabinofuranosidase to obtainspecific emulsification properties.

A very schematic drawing of a pectin 20 from coffee pulp is indicated inFIG. 2 a. MG refers to methyl group; AG refers to acetyl group, GUArefers to galacturonic acid, Gal refers to galactose, RHA refers torhamnose, and Ara refers to arabinose. Reference 21 refers to thehomogalacturonan region, reference 22 refers to a therhamanogalacturonan I region, and reference 23 refers to the neutralside chain region of pectins.

FIG. 2 b very schematically depicts a pectin obtainable with the processof the invention, wherein the pectins are cross-linked via cross-link(s)CL. Reference Fer refers to ferulic acid (a phenol that is the basis ofthe cross-links, together with the arabinose units), of which of eachpectin, via the arabinose units the pectins may be cross-linked with theaid of ferulic acid. The enzyme oxidoreductases, such as laccase and/orhorse radish peroxidase, may generate cross-links in the form ofpolyphenols, such as diphenols or even polyphenols having more than twophenol groups.

EXPERIMENTAL Example 1

Extraction and modification of pectin from coffee pulp Laboratory scaleprocedure:

Fresh pulp was obtained directly from a farm in Colombia in thebeginning of January 2012. The cherries were in optimum ripe state to beseparated from the bean in the wet mill. After the recollection the skinand pulp (exocarp and meso carp) were separated with a manual. The pulp,skin (pulp) are blended and freeze dried for transport to TheNetherlands. 1 g of freeze dried material is washed 3 times withacidified ethanol 80% and centrifuge at 3000 rpm for 5 min in each step,the solids are then suspended in water, the pH is adjusted to 2.0 withhydrochloric acid and fill to 50 ml volume. The suspension is shaken ina water bath at 70° C. for 3 h. The suspension is then centrifuged at3000 RPM for 10 min and the aqueous phase is separated from the solids.The solids are then neutralized and Sodium hydroxide is added to adjustpH at 10 and a final volume of 50 ml. The solids are suspended andshaken for 1 h at room temperature. After the alkali extraction thesuspension is centrifuged at 3000 RPM for 10 minutes and the aqueousphase is pooled with the acid solution. The pH is adjusted to 6.0 withdiluted alkali or acid. The spent material is then dried for furtheranalysis. The liquid obtained for extraction has a brownish colour. 3 mlof Hydrogen peroxide is (30%) is added to the liquor and 500 ul of horseradish peroxidase solution (5 mg/ml) is added and the solution isstirred for 24 hours at room temperature. After the incubation 4 volumesof absolute ethanol are added and the pectin is left to precipitate for2 hour at 4° C. After precipitation the suspension is filtered through aWhatman #3 filter paper with the aid of a Buchner funnel. The retenatesolids are washed with 100 ml of acetone and dried at 50° C. for 12 hwith high convection. The resulting film is then milled in a ball milland stored for further analysis.

Example 2

The following procedure for obtaining soluble polysaccharides fromcoffee pulp, with certain degree of polymerization here after calledpectin, this procedure is intended as a guide. Therefore modificationscan of course be chosen.

-   Materials: Coffee pulp (including coffee pectin): Coffee pulp from    the variety Coffea Arabica was obtained fresh (coffee cherries were    purchased directly from a farm in Colombia from the harvest of    December 2011); Peroxidase from horseradish: Peroxidase from    horseradish (HRP) was purchased from Sigma Aldrich EC 1.11.1.7 200    U/mg, Hydrochloric acid, sodium dihydrogen phosphate and sodium    hydroxide of analytical grade were purchased from Merck Dramstad    Germany.-   Method:The coffee cherries were washed with water and the pulp was    removed from the beans and parchments manually. The pulp was    immediately macerated in a conventional blender at high speed and    freeze-dried.

Clean Up Procedure (Washing Procedure):

1 g of coffee pulp was washed three times with a mixture of methanolwater 50/50 (%v/v) and centrifuge, the supernatants were pooled anddried under vacuum at low temperature the retentate goes to theextraction procedure.

Acid Extraction:

The retentate was then suspended in 35 ml of water, the pH of thesolution was adjusted to 2.0 by adding drop wise hydrochloric acid 6Mand the final volume adjust to 50 ml. The mix was left in a shakingwater bath at 90° C. for 1 h with continuous shaking and cool after toroom temperature with cold water. The suspension was centrifuge at 3500rpm.

Alkali Extraction:

The supernatant (first extraction product) is reserved (for laterapplication, see below) and the retentate (residual product of the firstextraction) is suspended in 25 ml of water. The pH is adjust to 12 withsodium hydroxide 4M drop wise, 5 ml of hydrogen peroxide solution 30% isadded, the final volume is adjusted to 50 ml and shaken for 1 h at roomtemperature. The suspension is centrifuged at 3500 rpm for 5 min and thesupernatant (second extraction product) pooled with the acid extractedfraction (first extraction product).

Enzymatic Reaction:

The pooled solution pH's is adjusted to 7.5 using diluted sodiumhydroxide. 5 mg of lyophilized enzyme(here laccase) is diluted in 1 mlof sodium dihydrogen phosphate buffer at pH 7.5 and added to the pooledsolution. The solution is left to react for 24 h at 35° C. temperatureand continuous stirring. After incubation, the solution is boiled in awater bath for ten minutes to inactivate the enzyme.

Precipitation and Drying:

To the final solution, 4 volumes of ethanol 96% are added and left forprecipitation for 3 h at 4° C., then filtrated through a Whatman N° 2cellulose filter paper in a Buchener funnel. The retentate is washed 3times with 50 ml of acetone, filtered and dried at 50° C. over 12 h. Theobtain pectin is milled in a Retsch ball mill for 3 minutes at maximumamplitude.

Cross linking of pectins through the ferulic groups which are attachedto the arabinan residues in the highly branched part of the pectinstructure of sugar beet pectins (SBP), is known in the art. However,this mechanism only explains the oxidative crosslinking of the hairyregion of the polysaccharide. Coffee pulp contains mainly galacturonicacid (GAU) (in between 60%-70% of the soluble sugars) and only 10% ofarabinan and only a small % of rhamnose. This leads to the conclusionthat coffee pectin (coffee pectin) is in fact predominantly composed byhomogalagturonan (HG) which is the smooth region of pectins, incomparison to SBP which is compose mostly of RGI which is the branchedregion of pectins. Therefore it is concluded that pectins from SBP andcoffee pectin are different in molecular structure, however coffeepectin shows an increased viscosity when sodium sulphite solutions areadded and also in high alkali conditions.

From laboratory analysis, it is known that there is a high amount ofpolyphenols attached to the cell wall material in coffee pulp. Itappears that there are at least two kinds of polyphenols; thepolyphenols bound to the cell wall polysaccharides and the free ones.The free ones are analysed as tannins and condensable tannins. The boundpolyphenols are hydrolysed by alkali in the second extraction step orstage, possibly because of the cleavage of the 2-O or 3-O bond in theGAU back bone chain. However there is a clear reaction when peroxidaseenzyme is added with the H₂O₂ substrate.

From this experiment is also possible to state that possibleapplications for the enzyme untreated pectin are also possible. In thiscase only alkali extraction will be needed to obtain a gelling pectinwhithout any other type of functionality. It is clear from the HPSEC(High Pressure Size Exclusion Chromatography) that unmodified coffeepectin has comparable size as the commercial citrus pectin (DM, degreeof methoxylation, 30%) despite the absence of gelling properties incoffee pectin. It is theorised that this is due to the high methyl andacetyl esterification pattern in the coffee pectin (DM 88%; DA, Degreeof amidation, 100%). The esterification pattern is too high to allowpolymer interactions in solution, and therefore nullifying theinteractions necessary to produce gel systems. Modification with pecticmethyl estereases enzymes could lead also to an increase of viscosity.In table 1a the sugar composition of the raw materials. (Rha: rhamnose,Ara: arabinose, Xyl: xylose, Man: mannose, Gal: galactose, Glc: glucose,GalA: galacturonic acid) is indicated in weight percentages.

Characterization of Pectin from Coffee Pulp Preliminary Results

TABLE 1a Sugar composition of the raw materials. (Rha: rhamnose, Ara:arabinose, Xyl: xylose, Man: mannose, Gal: galactose, Glc: glucose,GalA: galacturonic acid) Sugar composition % Rha Ara Xyl Man Gal GlcGalA Total sugar Coffee pulp Pectin 1 5 1 1 4 3 28.11 43 ParchementPectin 1 8 3 2 4 22 47.22 87 Raw pectin Extract 0 1 0 0 1 1 24.08 29

TABLE 1b Mole ratios for structure determination (UA: uronic acid)(Ara + (Ara + Sample name Rha/UA Gal)/Rha Ara/Gal Gal)/UA Gal/UA CoffeePulp pectin 0.03 10.84 1.47 0.37 0.15

TABLE 2 Degree of methylation and acetylation for pectin extrated fromcoffee pulp Degree of methylation/acetylation mg/mg mg/mg mg/mg SampleMetOH AcOH UA DM DA Coffee pulp pectin 4.85% 8.82% 0.26740 100% 97%

Coffee pulp present high contents of hydrolysable tannins, 3.17% in d.b.(dry basis) Tanins is a broad name for polyphenols attached to the cellwall material in plants, for coffee is present in the pectin structureas hydro cinnamic, chlorogenic, and caffeic acids which are polyphenolswith antioxidant activities.

There are examples in which polyphenols like ferulic acid, attach to thesoluble part of the cell wall material through the arabinose residues.As can be seen in tables 1a-2 arabinose is present in a significantpercentage in the coffee soluble pectin, and in the mucilage from theparchment fraction (parchment pectin). Therefore, the final pectin afterthe presently proposed process contains high amount of polyphenols. Theratio of phenolic groups to arabinose plus galactose will be between 20%to 60%, depending of the process yield. Commercial pectins that includephenolic groups are quiet rare, pectins extracted from sugar beet are anexample of this type of pectins, however, pectins from sugar beetpresent higher degree of branching and lower amounts of galacturonicacid which cause the low gelling properties. The other main differenceis the linearity of the coffee pectin molecule and molecular weight(>90,000 Da) which is close to the lemon pectin (120,000 Da to 160,000Da) and not to the sugar beet pectin (that is in the range 50,000 Da to80,000 Da), as can be seen in the HPSEC (high performance size exclusionchromatography) FIG. 3.

Also pectin extracted from coffee pulp with the present process,presents same digestion profile as the commercial pectin extracted fromcitrus peel when treated with endo-polygalacturonase (PG) as can be seenin the high performance anion exchange chromatogram (HPAEC) FIG. 4,while pectin from sugar beet is poorly digested by PG because thebranched structure. Other difference is the degree of acetylation andmethylation that can be tuned with the present process. However, the rawpectin from coffee pulp presents a unique high degree of acetylation andmethylation, this leads to the possibility to produce all range ofpectins with different degrees of methylation from 10% DM till as highas 80% DM this is unique for a commercial pectin as well.

Example 3 Experiment Data:

The following experiments were performed with freeze dried samples ofcoffee pulp (exocarp and mesocarp). The experimental design was asfollows: (1) Preparation of freeze dried coffee pulp (FD-coffee pectin):100 g of FD-coffee (i.e. freeze-dried coffee pulp) pectin was milled ina Retsch ball mill to a particle size that passes through a 6 mm mesh.(2) FD-coffee pectin was homogenized in demineralized water in one literof water; the pH was adjusted to low pH using Hydrochloric acid (HCl)37%. The acid was added drop wise. (3) The suspension was placed in awater bath at the correct temperature with continuous shaking for theexact period of time. (4) The suspension was then filtered hot throughcheese cloth; the cloth was squeezed by hand to remove most of theliquid from the solid mass. (5) The solid mass was then homogenized with0.5 l of water and the pH adjusted with NaOH 4M the alkali was addeddrop wise. (6) The suspension was placed in a refrigerated ice bath withcontinuous shaking for the exact period of time. (7) The suspension wasthen filtered in hot through cheese cloth; the cloth was squeezed byhand to remove most of the liquid from the solid mass. (8) Solutions aremixed together, 15 ml of Hydrogen Peroxide (H2O2) was added and thesolution is neutralized with NaOH or HCl. (9) 1.5 ml of horse radishperoxidase is added to the solution, and placed in a water bath at 40°C. for 12 hours with continuous shaking (10) 6 l of cold ethanol 90% isadded to the solution and left for 3 h at 4° C. The formed suspension isthen filtered through a Whatman 3 cellulose filter paper with the helpof vacuum. The retentate is washed with acetone several times to removeimpurities. (11) The retentate is dried in a convection oven for 12 hourat 50° C. and maximum air convection. (12) The dried coffee pulp pectin(coffee pectin) is weighed for yield and then milled for compositionanalysis.

Results (table 4): pH 1 Temp 1 Time 1 pH 2 Temp 2 Time 2 Yield coffee1.5 90° C. 30 m 8  4° C. 30 m 25% pectin001 coffee 2 90° C. 30 m 8  4°C. 30 m 23% pectin002 coffee 1.5 80° C. 120 m  8 25° C. 120 m  20%pectin003 coffee 2 80° C. 120 m  10 25° C. 120 m  30% pectin004 coffee 290° C. 30 m 13 25° C. 30 m  5% pectin005 coffee 2 90° C. 30 m 10 25° C.30 m 33% pectin006

Although yields of extractions were low in comparison to the amounts ofpectin reported in the literature, the produced pectin showedsurprisingly high amount of methyl and acetyl groups with DM of 80% andDA 100% while normal pectins after extraction do not present DM higherthan 75% or DA higher than 50%. Even though alkali hydrolysis is appliedin the extraction. Furthermore, the extracts from coffee pectin showgelling behaviour at high pH, this could be the reason why coffeepectin005 shows so low yield due to the fact that in the secondextraction, the gel is retained with the cellulose fraction. At this pHsubstantially all pectin may be hydrolized into its monomers wich willthen get into solution (when the precipitation occurs).

The laboratory size extraction of pectin from coffee pulp was initiallyperformed with freeze dried material. Since coffee pulp is produced ingreater ammounts that what can be freeze dried in an economical feasibleway, a method for avoiding coffee pulp oxidation and fermentation wasdeveloped. In a field experiment we used fresh coffee cherries, thefresh pulp was pressed to remove excess of water and the procedurefollowed was as substantially described above. In this example,preserved pulp was used instead of freeze-dried pulp. For thepreservation test we started with sodium bi-sulphite (NaHSO₃) which is acommon anti browning agent in the food industry and compared also low pHand heat treatment to inactivate the endogenous PPO (polyphenol oxidase)that causes oxidation in coffee pulp. The treatments were done in vitrousing cathecol as substrate wich is a common polyphenol present incoffee as well as in other plant materials, laccase was used as positivecontrol and a fresh coffee extract was use to oxidise the polyphenolsand record the change of absorbance.

After obtaining a satisfactory method for the preservation of the pulp,we proceeded to try the method at large scale (50 kg) of pulp collectedfrom the wet mills. This demonstrate the applicability of thepreservation method at large scales, which is needed to be able tocollect and preserve the pulp needed to make the process feasible. Forthe large scale extraction of coffee pulp we used wet preserved pulp (10kg) and scale it up trying to follow as much as possible the laboratoryprocess, we obtained low yields from this extraction.

Coffee is one of the most important agricultural commodities in theworld. Its most common cultivated varieties are Coffea arabica sp. andCoffea robusta sp. The total amount of green bean coffee produced in2011 was 7.8 million ton; demand is estimated to grow 10% in the nextdecade. Mechanization of coffee production has been carried out for thelast twenty years to cope with the demand, this has created newchallenges in the coffee chain. On one hand, mechanization increasescoffee productivity and lowers cost of production; on the other handmechanized production generates more agricultural waste and reduceslabour force in the plantation. Because of mechanization, coffeediscarded streams are being concentrated in the washing stations, wherethe coffee cherries are transformed into green beans. Today thetraditional wet method of coffee processing is being replace by the semiwet method or aquapulping. In the semi wet method the pulp is separatedfrom the beans by mechanical means, the mucilage is removed by frictionand mixed with the pulp. The discarded streams are mostly composed ofthe skin, pulp, mucilage and silver skin of coffee cherries. Thisdiscarded stream contains high amounts of polyphenols, polysaccharidesand sugars, as well as limited quantities of caffeine. The presence ofpolyphenols and their oxidised forms and caffeine makes the residueunfit for use as animal feed or as composting material. Thus, these sidestreams pose a major environmental problem in the coffee producingregions.

Coffee pulp represents about 40% of the total fresh weight of the coffeecherry. Traditionally, coffee pulp was used in small amounts asfertilizer and vermin-composting. However, these applications are nottechnically efficient for the high scale production of coffee. The pulpbiomass is rich in polyphenols, caffeine and complex polysaccharidessuch as pectin. These substances can be extracted from the pulp, byseparating and refining the products following the biorefinery approach.By separating the coffee pulp biomass the pollution loads could bereduced and the refined material could be transformed into valuablebiobased compounds, these can be used in the food and pharmaceuticalindustries.

Freeze-dried material showed stability in terms of oxidation andfermentation in comparison to fresh pulp. However, freeze-drying is anexpensive process, and is industrially of less interest for preservationof the increasing volumes of discarded biomass, on the coffeeplantations.

Although pectin occurs commonly in most of the plant tissues as acementing substance in the middle lamella and as a thickening on thecell wall, the number of sources that may be used for the commercialmanufacture of pectin is very limited. Because the ability of pectins toform gel depends on the molecular size and degree of degree ofesterification (DE), the pectins from different sources does not havethe same gelling ability due to variations in these parameters.Therefore, detection of large quantity of pectin in fruit alone is notitself enough to qualify that fruit as a source of commercial pectin. Atpresent citrus peels are the main sources of commercially acceptablepectins.

Commercial pectins are characterised by a high content of galacturonicacid, and this has become part of the legal definition for pectin usedas food additives or for pharmaceutical purposes. Typical requirementsare of a minimum of 65% of galacturonic acid on the ash andmoisture-free substances. Pectin normally comes from a range ofbotanically different tissues, which perhaps contain somewhat differentpectin structures. The traditional sources of pectin are apple pomaceand citrus peels, both coming from the left overs of the juice industry.Among the novel sources of pectin are sugar beets and sunflower heads,but these are not, at the moment commercially significant. Extraction ofpectins, as showed herein, may be by aqueous acid or alkline, especiallywith at least an acid extraction. The basic extraction process yields apectin of low degree of esterification (low DE pectin) as a result ofsaponification of the ester groups, whereas the acid extraction processgenerally yields a pectin of high degree of esterification (high DEpectin), approximately equal to the naturally occurring DE. High DEpectin has a degree of esterification of 50% or greater. Low DE and highDE pectin generally have different uses in foodstuffs, because they gelby different mechanisms. Both are sold commercially.

In the acid extraction process, plant material is treated with acid attemperatures especially between 70 and 90° C. for a time sufficient toremove desired amounts and quality of pectin from the cellulose plantmaterial. Extract juice from the extraction step is separated from thereaction mixture by filtration. Rotary drum vacuum filtration is commonin the industry because the cake is very mushy and difficult to handle.The pressed cake can be put through a re-extraction step to extract morepectin before being filtered and dispose of Pectin is precipitated fromextracted juice usually by alcohol precipitation (methyl, ethyl orisopropyl can be used). Salting out with aluminium chloride was used inthe past but new regulations do not allow such salt for pectins used inthe food or pharmaceutical industries (CODEX alimentarius). Theprecipitated pectin is separated from the precipitating solution byscreen filtration or other means; it is then washed, dried and milled tothe desired particle size. During processing the pectin may undergo anion exchange step to put it in the sodium form for ease of use infoodstuffs applications.

Other methods to extract pectin besides acid or alkali extraction havebeen used with the aim of reducing cost of operation and increase yieldsof extraction without loosing functionality of the pectin. High-pressuresystems could improve pectin yield without polymer chain degradation ofpectin, use of enzymes to degrade the cell wall, use of organic acids ornew filtration technologies such as cross flow filtration are promisingmethods to extract pectins in a more sustainable way.

Pectin differentiates not only from their natural source and type ofextraction process, different modifications of the pectin molecularstructure are possible yielding specific pectins with enhance or uniqueproperties that can be applied in the food, beverage and pharmaceuticalindustries. Pectins can be chemically de-esterified using acid, alkalior ammonia. Alternative to chemical methods de-esterification can bedone by enzymatic treatments. Other enzymatic modifications are possibledepending on the structure of the pectin molecule. It has been foundthat in sugar beet pectin the feruloyl groups esterified some neutralsugars in the side-chains of the so called “hairy regions”. It ispossible to take advantage of these feruloyl groups in pectin (beet). Itis also possible to cross-link the extracted feruloylated pectins bycarrying out coupling reactions. Such reactions can be achievedenzymatically by the use of e.g. a polyphenol oxidase, namely a Laccase.This oxidative cross linking of the pectins can add gelling propertiesto pectins which do not have such capacity. Because the gel is based onchemical bonds, it is also possible to recover the cross linked pectins,after drying. These modified pectins have remarkable water absorptioncapacities. Coffee pulp is rich in polysaccharides and may be apotential source of pectin. New methods for pectin modification couldunlock the potential of coffee pulp, as source of commercial pectin.Taking into account that coffee pulp is a polluting waste with a yearoutput of 20 million metric tons (estimations calculated from ICO), theimpacts of this technology could influence the pectin markets. To beable to extract pectins from coffee side streams, methods for preservingthe pulp need to be found and tested in the conditions of the coffeeplantations. As oxidation is the main post harvest process thattransforms this available biomass in an environmental problem in coffeeregions. Oxidation of the coffee pulp is mainly due to the enzymaticcatalysed reactions of polyphenols with endogenous peroxidases.Information on polyphenols from coffee pulp and polyphenols peroxidase(PPO) activity in the coffee fruit is scarce, and there is noinformation on how to preserve coffee pulp from oxidation. One of thereasons of this is that, coffee pulp is just a waste stream of thecoffee chain and no industrial alternatives are in place for itsvalorisation. The major problem for coffee pulp usage is the rapidoxidation that takes place after the milling operation. Although iscommon knowledge that oxidation happens in the coffee pulp, itsmechanism has not been understood yet. Part of the aim of this researchis to come up with a way to preserve the coffee pulp in such a way thatit could be used in a biorefinery scheme.

The laboratory scale extraction of pectin was done in a fieldexperiment. However, the raw material in this case is fresh coffeecherries. After testing the extraction from fresh material, we proceedto test chemical and physical treatments to stop oxidation of thebiomass. Once we obtained a satisfactory preservation process, we usedthe procedure to preserve a larger volume of fresh coffee pulp. With thepreserved biomass, we attempted the scaling up the extraction of pectin.We did composition analysis to the different fractions after extraction.A very schematic drawing of a coffe bean is indicated in FIG. 5 a, withcharacteristic contents in weight percentages, with M indicatingmucilage (4.9 wt. %), GB indicating the green bean (44.7 wt. %), Puindicating pulp (44.6 wt. %), and SS (p) indicating the silver skin(parchment) (5.8 wt. %). These weight percetages are characteristicvalues that may differe from bean to bean and from type of coffee beanto type of coffee bean.

Methods and Materials Laboratory Scale Extraction:

We extracted pectin from coffee pulp following the developed procedurein the laboratories with some modifications. Briefly, 10 g of fresh pulpwas collected and 50 ml of a Nitric acid solution 1% is added and mixed.We verified the pH to be between 2.0 and 2.5. The pulp is extracted for3 h at 90° C. with constant stirring. After extraction, the suspensionis cool down to room temperature and filtered through cheesecloth. Thepressed cake is then mixed with another 50 ml of Sodium hydroxide 0.1%and the pH adjusted to 9.0 with concentrated alkali. The suspension isthen stirred constantly for an hour at 10° C. We adjusted the pH againto 7 and filtered through cheesecloth, the solutions are then pooledtogether. The pH of the solution is adjusted (here to pH=3.5) by addingfew drops of concentrated NaOH or Nitric acid solution drop wise. Toclarify the solution, we centrifuged the pooled liquid at 2500 rpm for15 min. Ethanol 96% is added to the liquid in a ratio of three volumesof alcohol per one volume of pectin extract. We carefully mix thesolution and left it to precipitate for 12 h at room temperature. Wewashed the precipitated once with 100 ml of 96% ethanol and dried undercurrent of air for 12 hours. The dry pectin is dissolved in 100 ml ofphosphate buffer pH 6.0, the pH is verified and 0.1 ml of laccasesolution (18 mg protein per ml) is added. We let the solution react withcontinuous stirring for 12 h. The solution is then filtered through aWhatman No 2 paper. The clear solution is precipitated again with 0.31of 96% ethanol and dried under a current of air. 96% ethanol wasanalytical grade. Laccase was supplied as a freeze-dried powder. Wedispersed the Laccase in 0.01M phosphate buffer pH 6.0 and stored at 4°C. until use. The degree of esterification was measured using Megazymekit for pectin identification. Briefly, coffee pulp, sugar beet, lowmethyl esterified citrus, high methyl esterified citrus pectins and iotacarrageenan are dissolved in water and the pH is adjusted to 12 in orderto catalyse demethylation with production of polygalacturonic acidregions in the polymer. The pectate is incubated with pectate lyasewhich cleaves the polygalacturonic acid, releasing unsaturatedoligosaccharides which absorbs strongly at 235 nm.

Preservation Tests:

50 Kg of coffee cherries were collected in the region of CundinamarcaColombia in the month of May 2013. Handpicked ripe coffee cherries weretransported to a laboratory facility. Upon arrival, the cherries werefrozen immediately at −20° C. to avoid oxidation or fermentation. Thematerial was used in the following two weeks.

Laccase was kindly supplied in freeze-dried form. 1 g of powder had 18%protein. We dissolved the enzyme in 10 ml phosphate buffer solution atpH 6.0 and kept frozen in several vials. Each vial was 1 ml (18 mg ofprotein per ml) and when thawed was immediately used to avoid freezethawing cycles.

Cathecol was purchased from Panreac, sodium bisulphite, citric andnitric acid where analytical grade. Polyvinyl polyridone PVPP waspurchased from a local provider and was food grade quality. AMICON Spintube membranes MWCO 10.000 were acquired from Millipore corp. We carriedout all spectrophotometric measurement with a Pharo 100spectrophotometer (Merck Millipore).

The method for measurement of PPO activity in coffee pulp is in brief asfollows: 250 g of frozen coffee cherries were macerated and the beanwith mucilage and silver skin were separated from the pulp. The totalpulp obtained was 112.70 g in fresh basis (f.b.). This pulp was thenblended together using a hand mixer with 0.4 l of a 50 mM phosphatebuffer at pH 6.0. We left the blended mix to settle for 10 min andfiltered. We collected the supernatant, an aliquot of 10 ml of thesupernatant was taken, aprox. 0.1 g of commercial insoluble PVP wasadded. The mix was shaken for about a minute and then centrifuged for 15min at 10.000 RPM the mix was kept at 4° C. before analysis.

We took 3 ml of the supernatant, add it to an Amicon MCWC of 10.000 Damembrane and centrifuged for 30 min at 10.000 RPM. This procedure wasrepeated in the same membrane 3 times. With 1 ml of phosphate buffer 50mM pH 6.0, the remnant on the amicon membrane was wash three times,solubilised and reserved in an amber vial at 4° C. This is the enzymeextract of coffee pulp (EX). A solution of 60 mM of Cathecol wasprepared in a 50 mM phosphate buffer pH 6.0 and used as substrate. Formeasurement, we used a 1 cm light path length cuvette, 50 ul ofsubstrate and 50 ul of EX added to 2.9 ml of distilled water and theabsorbance followed in at 420 nm after 1 h, 2 h, 12 h and 24 h.

Large Scale Preservation and Extraction

We collected coffee pulp in two batches from two different types ofbeneficio (Post-harvesting process). The first 126 kg batch wascollected from a traditional wet mill operation. In the traditional wetprocessing method, cherries are transported with water through thepulping machine in a ratio of 4 litres of water per kilogram ofcherries. To remove excess of water the pulp was taken from the outputpipe of the pulping machine with a strainer. The pulp then was placed inhermetic barrels (3 barrels of 60 L each) which were filled 70% of thetotal volume with pulp. The barrels then were filled to the top with asolution of 1% of sodium bisulphite solution commercial grade, andsealed air tight for transportation. The ratio of solution to pulp wasabout 1 litre of solution per kilogram of fresh pulp.

The second batch of 96 Kg was collected from a semi wet method milloperation (Belcosub or aquapulping). In this method, a mechanical screwtransports the cherries through the pulping machine without water. Thenthe beans with the mucilage go through a scrubbing process, where themucilage is removed. The pulp and mucilage are mixed together, anddiscarded or composted. The pulp from this type of beneficio wascollected immediately after the milling operation by placing the barrelsin the pulp outlet. Since there was less water in the pulp than for thetraditional method, the barrels were filled just 60% of the volume, andthen filled to the top with a sodium bisulphite solution at 1%. Theratio of solution to pulp was higher due to the packing of the pulp inthe containers. The ratio was 0.81 of solution per kilogram of pulp. Thecoffee pulp collected and preserved in a sodium bisulphite solution wasperiodically checked for oxidation by change in absorbance of thesolution. For that, 100 ml of the solution in which the coffee pulp wassuspended was taken every 24 h, centrifuged and the absorbance of thesolution measured at 420 nm.

For the extraction of pectin from the preserved fresh pulp, we took 10kg of biomass taking care to remove excess water; we mixed the pulp with40 L of a 1% solution of nitric acid and blended with an industrial handmixer at maximum speed (18.000 RPM) for about 10 minutes. Once themixture was homogenized, we heated it with constant stirring to 92° C.for 3 h. After cooling down, we separated the solids with a strainer,and mixed with diatomaceous earths (DIE) as filter aid in a proportionof 1 kg of DIE per 10 L of liquid. We filtered the suspension using aframe and plate filter of 2.8 m² of surface. The membranes were of 100μm pore size constructed with nylon. The liquid is reserved and the cakeis pressed to remove excess of water. We mixed the residual cake with 40L of NaOH 0.5% and stirred constantly for one hour. The suspension wasthen neutralised (pH 7.0), the solids pressed and the liquid filteredusing the same press filter system and DIE. We mixed both alkali andacid extracts, adjusted the pH to 5.0, homogenized and filter it onemore time to obtain a clear solution.

The clarified liquid is the raw pectin liquor. This liquid was mixedwith one volume of ethanol 96% (Industrial grade) per volume of liquor.The pectin was left to precipitate for 12 h. The solution with the gelwas filtered using cheesecloth; the filtration bag was hoist to allowdripping of the residual ethanol solution for 24 h. The remaining gelwas dried at room temperature under a current of air (electric fan). Thedried pectin was dissolved in 5 L of a phosphate buffer solution 0.1Mand pH 6.0, then 5 ml of laccase (18 mg/ml) were added and the enzymewas left to incubate for 12 h. We mixed the modified pectin solutionwith one volume of ethanol 96% and let the modified pectin toprecipitate for 24 h. The suspension of pectin with ethanol is filteredthrough cheesecloth, pressed and dried under a flow of air at roomtemperature. FIG. 5 b shows the flow chart for a scale extraction of 10kg, with the following references.

TABLE 5 references of FIG. 5b Ref. Meaning 1.1 Preserved Pulp 10 g 1.2Nitric acid 1% 50 ml 1.3 NaOH 0.1% 50 ml 1.4 Homogenization 1.5 Acidextraction 1.6 T = 90° C. t = 3 h pH = 2.0 1.7 Filtration 1.8 Cake 1.9Filtrate 1.10 Alkali extraction 1.11 T = 10° C. t = 1 h pH = 9.0 1.12 pHAdjust 1.13 Filtration 1.14 Filtrate 1.15 Cake 1.16 Residue 1.17.Homogenization pH adjust 1.18 Alcohol precipitation 1.19 Filtration 1.20Washing 1.21 Resuspension 1.22 Enzymatic reaction 1.23 T = 20° C. t = 12h pH = 6.0 1.24 Alcohol precipitation 1.25 Filtration 1.26 Drying 1.27Modified Pectin 1.28 ETOH 96% 400 ml 1.29 Amonium Butter pH 6.0 50 ml1.30 Laccase 18 mg/ml 0.1 ml

Results and Discussion Laboratory Scale Extraction

For the laboratory size extraction, we used fresh coffee cherries, theextraction procedure is depicted in FIG. 5 b. The procedure differs fromthe one developed before in the (optional) press of the fresh pulp whilebefore we used freeze dried pulp. Coffee cherries have variations intheir composition not only between species (Arabica or Robusta) but alsobetween varieties. As most of the small coffee farms have a mixed ofvarieties, the composition can also change from farm to farm or harvestto harvest. The average composition of a coffee cherry is depicted in 5a. To start the extraction procedure the first step is to separate thepulp from the bean when we use the whole coffee cherry. In the coffeeplantations, this step is done mechanically by pressing the cherriesagainst a screen that remove pulp. Because in the traditional operationsthe cherries are transported with water, it is possible that the pulpsoaks water during the process. In the laboratory, the water content ofthe fresh coffee pulp was 73% of the total mass. But for preservedcoffee pulp in the sulphite solution, the water content was 80%. To havean average solid percentage of biomass for the extraction we firstpressed the pulp. The juice that came out was mixed with ethanol andsome small precipitate formed. After drying the precipitation weobtained 10 mg of dried mass, most probable the soluble free pectin. Thejuice as it is have low pectin content since the pectin is tightly boundto the cell wall, one of the reasons to avoid milling of the pulp beforepreservation is to reduce the loss of pectin in the pressing. From 100 gof pulp, 38.6 g of juice were squeezed out. The pressed pulp had a watercontent of 50%. When compared with the pulp from the semi-wet method thejuice squeezed from the same amount of pulp was only 20 g and the watercontent was 41%. Most probable the mucilage adds to the dry mass of thepulp. After obtaining the pulp we did a composition analysis. Table 6displays the results of the coffee pulp in dry basis.

TABLE 6 Coffee pulp composition in dry basis Protein (N*6.25) 8.2%Lipids 1.3% Fibre, crude 30.5% Ash 5.7% Carbohydrates 54.1%

After pressing, we took 10 g of pulp to proceed with the pectinextraction as described in the flow chart in FIG. 5 b. When we mixed thepulp with the acid solution the first change is the colour of the pulp.From the more brown yellowish colour due to the sulphite preservation,becomes a bright red colour. This is most probable due to the change incharge of the polyphenols and it will be discussed in the section ofpreservation. During acid extraction is evident how the pulp soakswater, if temperature is not controlled to avoid evaporation, then thesuspension becomes so thick that can burn. After the acid extraction thefiltration is one of the most critical steps in the process. The bestmethod until now, for filtration is through cheesecloth. Applyingpressure is the best way to remove as much as possible the liquid thathas been soaked by the biomass. Usually after pressing, the biomass hasbetween 10% and 15% of water. At this point the composition of theresidue was analysed, in Table 7 the results for the biomass in dry massafter acid hydrolysis.

TABLE 7 Residue of coffee pulp after acid hydrolysis and filtrationProtein (N*6.25) 16.7% Lipids 2.1% Fibre, crude 27.2% Soluble fibre12.1% Ash 10.5% Carbohydrates 40.2%

When suspended again in the alkali solution a rapid change of colour isvisible. The light brown to the very green dark of the pulp in this stepdepends much in the pH of the system. At pH under 8.0, the pulp is lightbrown and the viscosity of the biomass is appreciable. When the pH isset to 9.0, the whole mix becomes dark brown and the viscosity is lower(observation). One of the reasons to use an alkali and acid extractionis to achieve higher yields for pectin extraction. Table 7 shows thatthe acid residue still has a high content of sugars (carbohydrates).This makes evident that harsher conditions are needed to extract pectin.In other sources of pectin such as sugar beet pulp, many authors haveused alkaline treatments, the procedures comprises extraction andde-esterification in alkaline medium, followed by acid washing to removeashes and drying. Turquois et al demonstrated that under alkalineconditions, sugar beet pulp and potato pulp yields products containing ahigh content of pectic substances estimated on the basis of galacturonicacid contents and producing firm gels with calcium ions. As acid andalkali treatments give different properties and with the aim to maximizeyields both extractions are done in sequence. In Table 8the compositionof the biomass, after alkaline treatment of the acid residue. Bycomparing both table two and three, it is clear that the carbohydratesare lower after the alkali treatment. Moreover, the fibre andcarbohydrate contents are quiet close, this means that most of thepolysaccharides in the alkaline residue is composed by cellulose whichis the main component of the crude fibre. In both tables the content ofprotein is quiet high, residual nitrates from the acid may overestimatethe protein content.

TABLE 8 Composition in dry basis of the coffee pulp after sequentialextractions with acid and alkali (pH 9.0) Protein (N*6.25) 14.3% Lipids2.7% Fibre, crude 35.5% Ash 8.8% Carbohydrates 33.0%

After alkali extraction the filtrate is neutralized and both liquidspooled, again pH have an important effect in the behaviour of the pectinin solution. When pH is raised to 9.0 and then neutralized and combinewith the acid extract solution there is the formation of a instant gel.Whereas if pH is raised to 8.0, neutralized and pooled with the acidextract solution there is no gel formation. This may be a result of thede-esterification of the pectin molecule at high pH. This give a majorcontradiction, if coffee pectin extracted by alkali can form such gelswhen acidified, then the molecular size is big enough to form networksin water systems. This as a corollary tells us that the pectin extractedwith acid are different from the alkali extracted since acid pectins donot show calcium sensitivity or gelling behaviour before enzymaticmodification.

When pH is kept at 8.0 during alkali extraction, neutralized and pooledwith the acid extract the resulting liquid is homogeneous after mixing.To remove the polyphenols from the pectin is necessary to precipitatewith Ethanol the pectin and then washed several times. After washingwith solvent, the precipitate is disolved in Ammonium phosphate bufferpH6.0 0.1M. Laccase is added to the disolved pectin in a ratio of 0.01:1grams. After reaction for 24 hours with the enzyme, the solution shows acolour shift and higher viscosity (observation) compared with theoriginal solution.

Coffee pulp has an important content of polyphenols. We measured thecontent of polyphenols by the Folin Ciocalteau assay with tannic acid asstandard and the flavonols by the Vanilin method. The results for wholecoffee pulp is in Table 9.

TABLE 9 Content of phenol compounds and flavonols in coffee pulp Phenols1.8% Flavonoids 1.3%

Coffee pectin show abundant hairy regions and feruloyl groups. It ispossible that the reactions happening during the enzymatic modificationis in effect an oxidative cross linking of the coffee pectin. The colourof the solution changes also by the modification from light to dark. Thegel thus obtained needed several washing to remove the formed colourduring the incubation.

After modification, coffee pectin is precipitated with ethanol and driedunder a flow of air at room temperature. The yield from 10 g of coffeepulp is 25 mg of unrefined pectin. Table 10 shows the composition ofcoffee pectin. Table 11 presents the mass balance of the laboratoryscale extraction from dry freeze coffee pulp and fresh pulp in drybasis.

TABLE 10 Composition of coffee pectin after modification Protein 15.2%Soluble fibre 47.0% Insoluble fibre 0.4% Ash 10% Flavonols 2% Phenols 3%

TABLE 11 Mass balance for each extracted fraction from freeze-dried andfresh coffee pulp Fraction Freeze-dried pulp % Fresh pulp % Dry mass 9125 Carbohydrates 60 54 Acid extract 21 14 Alkali Extract 12 7 Residue 1320 Soluble sugars* 14 13 *Soluble sugars where calculated as thedifference of the yields with the carbohydrate content

To clean the pectin we had to wash several times with alcohol andmethanol, it was more difficult to clean the laccase-incubated pectinthan the extracted non modified pectin. After several washes withethanol and methanol (pure) we obtained a gel with a light pink colour.We collected the gel and dried at room temperature (20° C.) underflowing air for 24 h. After the gel was completely dried, we grinded itin a ceramic mortar by hand. This we denominated modified coffee pectin(MCP). The extract without modification was also washed with solvent anddried under the same conditions, this pectin was denominated coffeepectin (CP).

With the modified coffee pectin (MCP) we measured the content ofunsaturated uronic acid residues in the pectic polysaccharides. Theanalysis measured the activity of pectin lyase which cleaves the 1-4 α Dgalacturonic acid bonds. When these are unsaturated (no presence ofmethyl or acetyl groups) and by comparing with known pectin samples thecontent of unsaturated oligosaccharides can be extrapolated. The resultsare summarized in Table 12 below:

TABLE 12 Analysis of the concentration of unsaturated pectin accordingto JECFA (Joint FAO/WHO Expert Committee on Food Additives)Polysaccharide Unsaturated product X 10⁻⁴ M HECP (high ester citruspectin) 3.10 SBP (sugar beet pectin) 2.03 MCP (modified coffee pectin)0.285 CP (coffe pectin) 0.476 Carrageenan 0.004 Pectin molecular weightis calculated as polygalacturonic acid with extinsion coefficient of4600 M⁻¹ cm⁻¹

These results show the difference between sugar beet pectin (SBP), MCPand high ester citrus pectin (HECP). The lower the value in the table,the less free places the polysaccharide has to form hydrogen bonding orto interact with ions. This can explain why CP (coffee pectin) and MCP(modified coffee pectin) do not gel and therefore it opens thepossibility to use enzymes like pectin methyl estereases (PM) or alkalitreatments to de-methylate the polysaccharide. Other importantcharacteristic of MCP is that its unsaturation is lower than SBP, thisof course shows that although both are pectins, the products in itselfare molecularly different. Furthermore, it shows that MCP has a morehydrophobic nature than HECP or SBP and if you add the content ofprotein, its application as food additives could be clearly inemulsification, but also in other completely new applications. Hence,this identification test indicates qualitativeley the differerencesources or type of pectin. It shows that coffee pectin is indeeddifferent from Citrus pectin and SBP. This is unique for thehydrocolloids soluble in water. For the analysis of table 12, theMegazyme kit for pectin identification was used, according to the Assayprocedure K-PECID 11/11 (Megazime International Ireland 2011).

Preservation

To understand the oxidation development of phenolic compounds present incoffee pulp, it was necessary to extract the enzyme(s) that catalyse thereaction while avoiding the oxidation of such compounds. In previousliterature authors haveextracted and characterized the enzymesresponsible for oxidation in the coffee leaves and endosperm (beans).They found that the enzymes responsible for the oxidation are from thefamily of polyphenyl peroxidases PPO (E.C. 1.10.3.2). These types ofenzymes contains copper in their active sites and are responsible forthe hydroxylation of monophenols to o-diphenols and the sub sequentialoxidation from o-diphenols to o-diquinones. PPO enzymes are responsiblefor the enzymatic browning in most fruits and plants. Browning is theresults from both enzymatic and non-enzymatic oxidation of phenoliccompounds. The initial oxidation products are quinones, which rapidlycondense to produce relatively insoluble brown compounds (melanin) Themost important factors that determines the rate of enzymatic browning offruits and vegetables are the concentration of both PPO and phenoliccompounds presents, the pH, the temperature and the oxygen availabilityon the plant tissue

PPO enzymes from coffee showed two distinctive bands in a SDS-PAGE,these bands are in 64 and 29 kDa of size. The purified PPO showed higheraffinity with chlorogenic acid (Km=0.14 mM) DOPA (Km=1.36 mM), cathecol(Km=4.75 mM) and pyrogallic acid (Km=6.16 mM) respectively. Although itwas expected that chlorogenic acid has more affinity to PPO, theoxidation of this compound proceeds faster than other phenols such ascathecol. It was decided to use cathecol to have a better control inoxidation process, also the colour formation of cathecol has been usedby other authors due to its increase in absorbance at 420 nm whenoxidized. We decided to extract all enzymes complex of the pulp andfollow the oxidation of cathecol as substrate using the enzyme extract.When subjected to concentration of the enzymes from the coffee pulp itis important to mentioned that the retentate in the Amicon membrane wasa dark brown colour. In the extraction PVPP was used to bound mono anddiphenols that could be in solution. After centrifugation, it wasvisible that many of the colour compounds in the coffee pulp extract areretained, this could mean that polyphenols are bound to bigger moleculeslike proteins and polysaccharides. The coloured compounds did not elutethrough the membrane which should let pass molecules smaller than 10kDa. Because the objective was to test the inhibition of the enzyme(s)by addition of chemicals (sodium bisulphite) or by temperaturetreatments in processing circumstances, we kept all possible enzymesizes that can have an effect in the oxidation of polyphenoliccompounds. The extract of the enzyme was used as it was extracted fromthe Amicon membrane, and suspended in 50 mM phosphate buffer at pH 6.

Inhibition In Vitro:

To calculate the oxidation inhibition, the change in absorbance after 24h of the solution with enzyme and substrate was compared, with thechange of absorbance of the samples with sodium bisulphite solution orwhen temperature was applied. For the inhibition with sodium bisulphite,there was a decrease in the absorbance of the enzyme extract solutionwithout added cathecol. There is a clear interaction of the bisulphitesolution with the coloured compounds in the coffee enzyme extract. Thisalso reinforces the idea that polyphenols could be part of biggermolecules that were not eluted through the Amicon membrane. Or thepolyphenols themselves are polymers bigger than 10 kDa. Interactions ofproteins and polysaccharides with polyphenols such as resorcinol andcathecol have been studied in the past. The interactions of bovine serumalbumin (BSA) with cathecol show that complexation is reversible, and isdriven by nonspecific surface phenomena. This complexation occurs viaboth hydrogen bonding of the polyphenol to the exterior ketoimide, andpolar groups on the protein and hydrophobic interactions. Thesemechanisms may be dependant of the protein concentration and the pH ofthe. Polyphenols concentrations in coffee pulp are high. Protein wasalso analysed in the fresh coffee pulp and it was found to be 8% (d.b.)(Table 6). If proteins from the same coffee extract go into solutionwhen blending, the polyphenols could bind to the proteins. Because theextraction takes place at a pH where the binding of polyphenols toproteins such as BSA have been found to be favourable, the same bindingprocess could happen with the proteins from the coffee pulp.

The sample subjected to temperature without substrate showed an increasein absorbance, in comparison to the same sample without temperaturetreatment. This was expected since temperature increases the rate atwhich oxidation occurs. In the sample with only substrate, there wasalso an increase in absorbance. The inhibition percentage was correctedfor those changes by subtracting the absorbance of the enzyme solutionwithout substrate, which as can be observed in Table 13 has higherabsorbance after 12 h of incubation at room temperature (20° C.).

TABLE 13 Light absorbance measured at 420 nm after 12 h for theoxidation of cathecol (substrate) Substrate blank Enzyme Oxidation (AU(absorbance extract reaction Oxidation Treatment units)) blank (AU) (AU)Inhibition % None 0.072 0.158 0.385 0% Sodium 0.063 0.095 0.083 105%bisulphite Temperature 0.137 0.171 0.184 94%

Sulphite compounds can be used to avoid oxidation in foodstuff. As weshowed in FIG. 5 c, the coffee enzyme extract oxidizes cathecol, but inthe presence of sodium sulphite the oxidation is stopped as is showed inTable 13. The enzyme extract is a mix of naturally occuring endogenousenzymes; PPO is present in this exctract. The oxidation of cathecolshows that the main reaction contributing to browning of the coffee pulpis the oxidation catalysed by the PPO.

Temperature is another way to stop browning of fruits and vegetables.PPO may be inactivated when subjected to temperatures over 40° C. As themajority of processes for thermal inactivation are intended for freshfruits and vegetables, it is desirable to expose the product as less aspossible to higher temperatures in order to preserve appearance of theproduct. In the case of coffee pulp, the inhibition of the enzymaticbrowning is desired for preservation of the biomass so posteriorprocesses can be applied. As appearance or texture of the product is notof importance, coffee pulp can be subjected to higher temperatures andlonger times. However, the impact of such treatments on the finalquality of pectin has to be assessed. We decided to heat the coffeeenzyme extract for 30 min and 90° C. so to mimic a normal blanching stepfor the pulp, in such way not only PPO but other enzymes as well asfungal activity will be stopped. As can be seen in table 13, temperaturealso inhibits enzymatic browning, however it also catalyzed oxidationtherefore the change in absorbance between the substrate blank, theenzyme blank and the oxidation sample. The higher absorbance compared tothe sulphite could be due to the non enzymatic oxidation of thepolyphenols present in the coffee extract as well as the oxidation ofcathecol by the presence of oxygen. In any case, temperature could beapplied in coffee pulp to stop enzymatic browning.

Large-Scale Preservation of Coffee Pulp:

As sulphite solution gave positive results for inhibition of oxidation,we use a 1% solution of sodium bisulphite to submerge the pulp collectedfrom the wet mills.

In the first 24 h there was a change in the pulp's colour from a darkbrown to more clear yellow. Nevertheless, there was no change in theabsorbance at any point at 420 nm. The clear yellow colour of thesolution was stable during the rest of the analysis (15 days) afterwhich there was clear evidence of fermentation in the container. It isimportant also to mention that in the first 3 days of the assay thecoffee pulp that was in the surface and not immersed in the Sodiumbisulphite solution, changed colour (dark brown pigment), and wasattacked by fungi (presumably white rot by microbiological analysis, notshown). However, all the rest of the pulp which was submerged in thesolution continued to lose pigmentation (from the red colour of thecoffee cherries to yellow colour of the solution) and no attack of fungiwas registered. Therefore, to continue the experiments the layer ofoxidized pulp was removed and weight was applied to the biomass to keepit submerged in the solution.

The use of the sulphite solution solves a great logistical problem sincein this solution the coffee pulp can be preserved for longer times.However, after some time (20 days) The pulped that was preserved in thesulphite solution started to ferment. To keep the integrity of thepectin fermentation has to be stopped; a better preservation form has tobe implemented. Drying of the pulp could solve the problem offermentation and reduce transportation costs. Possible dryingtechnologies that can be applied to coffee pulp based on technical andeconomic factors may include one or more of: batch drying, sun drying,tunnel driers, belt driers and rotary driers. For batch driers one ofthe limiting factors is the compressibility of coffee pulp. For theother four technologies the capacities are calculated according to theproduction of pulp for a medium size wet mill per day. At the peak ofthe season a medium size wet mill can produce 63.50 metric tons of pulpper day, the authors use the maximum content of water that the pulp havewhich is 85%. Therefore is necessary to remove 52.16 cubic meters ofwater in 8 h to obtain coffee pulp with 10% moisture content.

Drying will be a mayor cost for the use of coffee pulp biomass. At themoment coffee pulp is been dried under constant airflow close to a sundry system, this takes around 8 days with a load of 20 Kg per squaremeter. We have found that after blanching and pressing the pulp thistime is reduced to 2 days at the same conditions.

When the pulp is stored in the sulphite solution the biomass can betransported, when drain and press, The pulp do not show oxidation. Afterdraining, the pulp was placed in trays for sun drying, after one day thepulp showed fungal growth. We checked the pH of the biomass and found itto be 6.0. This pH is optimal for fungi, this is why we decided toadjust the pH of the biomass to 3.5 just before blanching. The result isthat the biomass turn colour to bright red, and after several days ofdrying there is no evidence of fungal growth or oxidation. However whenwe press the pulp we are losing soluble pectins, taken into account thatblanching at low pH could lead to hydrolysis, preserving and drying thepulp could lead to low yields of pectin. The flow chart for thepreservation of coffee pulp at large scale is as follows in FIG. 5 dwith the following references.

TABLE 14 references of FIG. 5d Ref. Meaning 2.1 Coffee cherry 100 Kg 2.2Losses 1.26 Kg 2.3 Depulping 2.4 Pulp 44.84 Kg 2.5 Mucilage + parchment56.15 Kg 2.6 Demucilaginator 2.7 Dry coffee parchment 25.02 Kg 2.8Drying 2.9 Water 22.96 Kg 2.10 Mucilage 8.17 Kg 2.11 Sulphite soakingStorage 2.12 pH Adjust 2.13 Blanching 2.14 Press 2.15 Pressed pulp 27 Kg2.16 Drying 2.17. Dried preserved pulp 10 Kg 2.18 NaHSO3 1% 44.84 L 2.19Nitric Acid 20% 0.22 L 2.20 Juice 71 L 2.21 Water 17 Kg

The preservation procedure allows to keep pulp stable for longerperiods, although its stability and the impact on pectin quality havenot been assessed. We already extracted pectin from preserved materialswith similar results as the freeze dried one. We have not tested theperformance of the pectin in any applications yet.

Large Scale Extraction of Pectin from Coffee Pulp

The first problem when scaling the process with wet preserved pulp isthe homogenization of the biomass with the acid. Blending the biomassgives a coarse mix, to increase yields is necessary to reduce theparticle size of the pulp. We tried to use a rudimentary extruder toachieve the milling of the pulp. The results were not good since thepulp blocks the extruder die very fast. The water holding capacity ofthe biomass probably causes this problem. So the only way to achieve agood mix was with an immersion blender. However, the milling of the pulphas to be optimized in the future for larger extractions or volumes.

For the first large scale extraction our starting material was the pulpcollected from the traditional wet milling process. The pulp waspreserved in the sulphite solution (1%), and no oxidation was visible.We took the pulp with a strainer and press out enough of the water inorder to maximize the amount of solids. The water leached from the pulpshowed turbidity. This could be pectins and other soluble material thatare in suspension, because during transport we had some spillage ofwater it was not possible to do a mas balance on the liquid itself.Though, it was visible that the pulp had soaked in sulphite solution.When the drained pulp was mixed with the acid solution, the colour ofthe mix turned to red. As discuss before this is probably the effect ofpH change of the polyphenols. When heated the sludge separates very fastso constant mixing was needed to keep the biomass in contact with theacid solution.

The solution had a bright strawberry-red colour, but the viscosity wasnot high. However, the load of suspended solids was. The brix of thesolution was 15°. Calculation of the brix by refractometry was notaccurate, due to the precipitation of some of the solids over time. Itwas clear that there were fine particles suspended in the solution, thisfouled very fast any cheesecloth or filter paper that we tried. To get aclear solution the particles had to be removed. At laboratory scale, weremoved all solids by filtration through a paper filter Whatman #2 (100um) by vacuum filtration or with a cheesecloth. At large scale thecheesecloth was inefficient and we lost a lot of material.

With the amount of solution to be filtered, an efficient process had tobe used to get rid of the particles. We opted for a traditional platepress filter of 20 cm×20 cm and seven frames (0.28 m² filtration area,Tellarini pompe Italy). Initially we used the same Whatman #2 paper asmembranes, which we also used in the laboratory. Unfortunately, theresult was very low, since the filter paper got blocked almostimmediately (fouling). To make filtration possible we designed syntheticfabric membranes of 100 um particle diameter custom made to the size ofthe filter press. With the newly made membranes, the filtrationimproved, but still the operation cycle was too short with less than onelitre of solution per cycle. The membrane manufacturers then recommendus to use a filtering aid, in this case diatomaceous earths (DIE) wasthe best performing material. So we mixed the solution with 1 Kg of(DIE) for every 10 l of solution. From less than a litre per cycle, weincreased the efficiency to seven litres per cycle. The suspendedparticles stick to the DIE and form a filtration bed in the membrane.After the cycle, the formed cake can be washed for the recovery of thesuspended particles and the DIE. We recovered a portion of the suspendedparticles and did a composition analysis. The particles are composed by23% protein and 51% carbohydrates, these particles could be of interestin the future.

After filtration, the brix content of the solution was 10°. Then weadded ethanol in a ratio to the solution of 4:1. We measured thepercentage of ethanol with an alcoholimeter (percentage in v/v) andfound out the solution was over the recommended value. This means wecould lower the amount of ethanol for effectively do the precipitation,in the next batches we corrected this value to achieve only 70%, thecorrect amount of ethanol was 1:1 ratio v/v.

After fixing the ethanol concentration and the filtration problems weproceed to a big scale extraction following the flow chart process inFIG. 5 e with the following references:

TABLE 15 references of FIG. 5e Ref. Meaning 3.1 Preserved pulp wet 10 Kg3.2 Homogenization 3.3 Acid extraction 3.4 Press 3.5 T = 92° C. t = 3 hpH = 2.0 3.6 Cake 20 Kg 3.7 Alkali extraction 3.8 pH Adjust 3.9 DrainPress 3.10 Filtration 3.11 Suspended solids 34 Kg 3.12 NaOH 0.5% 40 Kg3.13 Drying 3.14 Solids 2 Kg 3.15 Cake 4 Kg 3.16 DIE 4 Kg 3.17 T = 10°C. t = 1 h pH = 9.0 3.18 26 L Liquid 3.19 30 L Liquid 3.20 Nitric acid1% 40 Kg 3.21 DIE 4 Kg 3.22 Filtration 3.23 Suspended solids 20 Kg 3.2414 L 3.25 Homogenization pH adjust 3.26 Alcohol precipitation 3.27ETOH96% 31.2 Kg 3.28 Filtration 3.29 EOH 70% + Sol. 65.2 Kg 3.30 6 Kg3.31 Homogenization 3.32 Amonium Buffer pH 6.0 3.33 Enzymatic reaction3.34 T = 20° C. t = 12 h pH = 6.0 3.35 Laccase 18 mg/ml 1 ml 3.36Alcohol precipitation 3.37 ETOH 96% 3.9 Kg 3.38 Filtration 3.39 ETOH70% + Solubles 9.9 Kg 3.40 Solids 5 Kg 3.41 Drying 3.42 Modified Pectin170 g

With the information in the mass balance, we calculated the yield ofextraction from wet preserved pulp, taking into account that the wetpulp is 75% moisture, the total solid content is just 2.5 Kg. Afterdrying the final gel, the pectin in mass was just 170 g. This give usonly a 6.8% of yield. By revising the process, the most clear losses ofpolysaccharides could be in the filtration steps. The suspended solidshave a high content of protein and carbohydrates as mentioned before.However, these particulates do not solubilise at pH between 3.5 and 7.0which is the pH range for pectin stabilization. Other factor that lowersthe yield is the extraction of the soluble solids. In the laboratory wemeasure the content of water soluble but alcohol insoluble solids(Soluble fibretable 7), this analytical method makes correction forprotein content (AOAC 985.29 18th edition) and its value is a goodapproximation of the content of pectic material. The 170 g of pectinyield from the 10 Kg of wet pulp (6.8% in dry basis) is less than halfof soluble dietary fibre (12%) in the coffee pulp. Also is important tomention that the colour of the pectin extracted is very dark, whichcould indicate the presence of flavonols which reduce the content ofsugars in the total mass. Much work is still to be done in theextraction and purification of pectin from coffee pulp.

The reproducibility of the laboratory method for extracting coffee pulppectin was tested and the results agrees with the previous extractions.Though the method is reproducible, there still much to improve in theextraction parameters and filtration techniques to increase yields, newmethods for pectin extraction use in other plant materials like steamexplosion, microwave or high pressure extractions could improve theyields of pectin. Other aspect to research further is the purificationof the pectin. Since coffee pulp have high quantity of polyphenols, isnecessary to find a way to remove them so the pectin can be used in foodapplications without given colour shifts.

To avoid the rapid oxidation of coffee pulp, we can use sodium sulphitesolution to collect and store the biomass. The sulphite solution showsan anti-oxidative effect when applied to the coffee pulp. This methodcan be scaled-up quickly and seems to be cost effective for largerquantities of biomass. We also blanched and dried the biomass to avoidfermentation. After drying, the pulp is stable and could be shippedeverywhere where the extraction process is implemented. The quality andapplicability of the coffee pectin extracted from preserved and driedpulp should be tested in the future to confirm the usefulness of thismethod.

In the scaling up of the process we encounter several hurdles that needattention in future optimizations. One of the main issues is the lowyields of pectin that we are obtaining from the fresh (preserved) coffeepulp. Also there is many technical problems in the filtration methodswhere losses are important. Finally, more attention has to be put inpurification of the extracted pectin. The main focus right now should beto find optimal processes for extraction and purification avoiding theuse of solvents.

The gelling properties of the coffee pectin obtained with the presentinvention are surprisingly good and were compacted with other gellants.The gelling properties seem to be even better than arabic gum, whencompared at the same concentration. Likewise, also, the gellingproperties of the coffee pectin as described herein, are substantiallybetter of sugar beet pectin (at the same concentration). It furtherappears that without the enzymatic treatment of the present invention, afurther enzymatic modification of the coffee pectin is very difficult oreven impossible. Hence, surprisingly an enzymatic modification of coffeepectin per se is very difficult or even impossible, whereas for otherpectins this is no problem. Only after application of the process of theinvention, including acid extraction and enzymatic modification, furtherenzymatic modification with other enzymes is possible.

Different sources of pectins have different molecular characteristicsand different degree of methylation, or acetylation. As far as the priorart is concerned, only pectins extracted from sugar beet may showferuloyl esters attached to the pectin structure. However, in thepresent invention it was surprisingly found that pectin extracted withthe herein described procedure from coffee pulp shows presence ofphenolic compounds (ferulic acid is phenolic compound also known ascinnamic acids). Moreover, the phenolic compounds are bound to thehigher molecular weight fractions of the acid soluble, alcohol insolublecell wall material of coffee pulp. Only pectins with feruloyl groups canbe cross-linked by enzymatic means using enzymes such as laccaseaccording to the present process. Citrus pectin or apple pectin do notshow feruloyl groups attached to the neutral branches of the molecule. Apectin molecule can be seen as a backbone of galacturonic acid (smoothregion or HG) attached to ramified structure comprising rhamnose,arabinose and galactose unit is (hairy region or RG). Methyl groups andacetyl groups are attached to the galacturonic acid fractions, whileferuloyl groups are found only in the neutral side chains. At higher pH,the methyl groups may be hydrolysed from the pectin backbone. The acetylgroups need harsh conditions to be hydrolysed (e.g. higher pH and highertemperature). At high pH, pectin structure is broken down throughβ-elimination mechanism, which only affects the galacturonic fractionsthat do not have other side groups. In summary at high pH, methyl groupsare hydrolysed and the smooth region is broken in the sites where thereare no other side groups. When the enzymatic modification occurs, it mayespecially happen through the neutral side chains in the RG region. Thatgives a new molecule with lower DM and higher DA. Enzymaticcross-linking of pectins can only occur where feruloyl groups arepresent in the molecule. From literature we know that only sugar beetpectin and now coffee pectin present this type of groups in their pectinstructure. The cross linking reaction is catalysed specifically by forinstance PPO (polyphenyl peroxidase) which horse radish peroxidase andlaccase are part of. Therefore the technology is based in both the typeof enzyme used and the source of the pectin. Sugar beet pectin structureshows an RGI type configuration in which RG represents in between 49 to59 mg/g of dry material and a maximum of 656 mg/g of dry material ofgalacturonic acid when de-esterified using plant PME, while incomparison with coffee pectin RG represents only 10 mg/g dry matter andgalacturonic acid represents 281 mg/g of dry matter. Hence, coffeepectin is substantially not similar to sugar beet pectin in structure.Further, coffee pectin has a higher degree of acetylation in comparisonto red beet pectin. In the process to obtain coffee pectin concentrationof polyphenols in both extracts are very high, it has been proven atlaboratory scale that without any treatment for the reduction of activepolyphenols in the pectin extract, substantially no enzymatic reactionwill take place. The only reaction that is carried without anyrestriction is the oxidative cross linking of the pectin. So to be ableto modify enzymatically pectin in a coffee matrix (pectin from coffee)is to remove amongst other the tannins or make them react as describedherein. Without laccase and/or other oxidoreductase it may not bepossible to do other enzymatic reactions on coffee pectin.

As experimental conclusion coffee pectin can be extracted from coffeepulp with good yields. Moreover, coffee pectin can be chemically orenzymatically modified to produce pectins with acceptable gellingproperties. The polyphenols in the extracted pectin show an (optical)absorbance wavelength shift with increasing pH, and this stronglyindicates the presence of polyphenols in complex networks likeflavonoids. Therefore possible feruloy esterified neutral side chainsare plausible in the structure.

Commercially available pectins are characterized by a high content ofpolygalacturonic acid, the legal definition for pectin used as a foodadditives or for pharmaceutical purposes requires that at least 65% ofthe ash and moisture free content be galacturonic acid. Thisrequirements limits the potential sources of food and pharmaceuticalpectins. The difference in galacturonic acid between pectin sources islinked mostly to the difference in concentration of the neutral sugarsthat are part of the molecule, largely arabinanas and galactan moieties.

The best know property of pectin is that it forms gels with sugar andacid. This can be seen as a partial dehydration of the pectin moleculeto a degree where it is intermediate between solution and precipitation.The particular structure of pectin imposes some specific constrains.High methoxyl pectin, unlike alginate, does not contain sufficient acidgroups to gel or precipitate with calcium ions. At a pH well above thepK value for the acid groups, the molecule possesses sufficient negativecharge to prevent gelation under practical conditions in sugar watersystems. As the pH is gradually reduced, the pectin is capable offorming a gel at first at high sugar contents (around 80% in brix scale)and at gradually lower sugar contents as the pH is reduced.

One of the commercial variables of pectin structure is the acetylationand methylation pattern due to the relation of this value with thegelation behaviour. Galacturonic acid in the polymerized form, has thepossibility to show methyl groups attached to the carboxylic groups. Themethylation percentage is taken as the ratio of methyl groups per moleof galacturonic acid present in the pectin. Indeed one of the commercialcharacteristics of pectin is its degree of methylation DM. The degree ofmethylation separates between rapid set pectin and slow set pectin. AtpH values well below 3.0 a very rapid setting pectin with degree ofesterification of above 72% will form a gel with 55% or somewhat less ofsugar. Slow set pectins are produce by mild hydrolysis of the estergroups to a degree between 58%-65% DM, and hence bear more charge at agiven pH. In consequence the gel strength and setting temperature curvesare displaced to lower pH. These pectins are used where a lower settingtemperature is required, or where the rate of set would otherwise be toohigh because of the increased sugar solids of the product. Low methoxylpectins are produced by de-esterification to a point where less than 50%of the total carboxyl groups are esterified. If this process is carriedout using acid or alkali, the balance exists as free acid groups; thesepectins are termed conventional or non amidated low methoxyl pectins.Alternatively, pectin may be reacted with ammonia, usually by aheterogeneous reaction in an alcohol suspension. This reaction producesamidated pectin containing acid amide groups in addition to acid estergroups. Both types of low methoxyl pectins are believed to gel in anegg-box mechanism with calcium ions.

In conclusion, pectins that can be modified by enzymes to meet specificdegrees of methylation and acetylation like the coffee pectin describedhere, allow to meet different types of setting properties as well asdifferent behaviours when gelling, therefore the pectin obtained withthis process can be tailored for different applications.

Hence, the invention also provides a process for producing a pectinbased product comprising using the polyphenol functionalized coffeepectin extract obtainable by the process as defined herein (and/)or thepolyphenol functionalized coffee pectin extract ase defined herein andprocessing the polyphenol functionalized coffee pectin extract togetherwith one or more other components into the pectin based product. Forinstance, the pectin based product may comprise a food product. Inanother embodiment, the pectin based product comprises a pharmaceuticalproduct. In yet another embodiment, the pectin based product comprises aneutraceutical product. The term “pectin based product” may relate toany product comprising the polyphenol functionalized coffee pectinextract, even when the amount is low. The one or more other componentsmay be any other component necessary to make such food product,pharmaceutical product or neutracutical product, respectively.

1. A coffee pulp treatment process comprising: a. Providing coffee pulp,obtainable from a production process for producing green coffee beansfrom coffee cherries; b. Extracting from the coffee pulp a pectincomprising extract, wherein extraction is performed under acidconditions, to provide the pectin comprising extract; c. Enzymatictreatment of the pectin comprising extract, wherein the enzymatictreatment comprises a treatment with one or more enzymes selected fromthe group consisting of an esterase and a reductase, to provide aenzymatically treated pectin material, wherein the enzymatic treatmentcomprises at least a treatment with an oxidoreductase; and d. Extractionof polyphenol functionalized coffee pectin extract from theenzymatically treated pectin material.
 2. The process according to claim1, wherein the coffee pulp is subjected to a first extraction under acidconditions, leading to a first extraction product and a residualproduct, wherein the residual product is further subjected to a secondextraction under alkaline conditions, leading to a second extractionproduct and a second residual product, and wherein the from this secondextraction obtained second extraction product is recombined with theremaining first extraction product from the first extraction, andwherein these combined pectin extraction products are then furthersubjected to the enzymatic treatment.
 3. The process according to claim1, wherein the enzymatic treatment comprises at least a treatment withone or more enzymes selected from the group consisting of EC 1.10 or EC1.11.
 4. The process according to claim 1, wherein the acid conditionsof the first extraction are at a pH in the range of 0.5-4, especially1.5-3, wherein the first extraction is performed at a temperature of atleast 80° C., wherein the alkaline conditions on the second extractionare at a pH in the range of 8-14, especially 9-11, and wherein the oneor more enzymes are selected from the group consisting of diphenoloxidoreductase, peroxidase, laccase, pectin-esterase, methyl-esterase,poly galacturanase, endo polyglucanase, and exo polyglucanase.
 5. Theprocess according to claim 1, wherein the coffee pulp is subjected to afirst extraction under alkaline conditions, leading to a firstextraction product and a residual product, wherein the residual productis further subjected to a second extraction under acid conditions,leading to a second extraction product and a second residual product,and wherein the from this second extraction obtained second extractionproduct is recombined with the first extraction product from the firstextraction, and wherein these combined pectin extraction products arethen further subjected to the enzymatic treatment.
 6. The processaccording to claim 5, wherein in the first extraction a first extractionliquid is applied that comprises H₂O₂, wherein the alkaline conditionson the first extraction are at a pH in the range of 8-14, especially9-11, wherein the acid conditions of the second extraction are at a pHin the range of 0.5-4, especially 1.5-3, wherein the second extractionis performed at a temperature of at least 80° C., and wherein the one ormore enzymes are selected from the group consisting of diphenoloxidoreductase, peroxidase, laccase, pectin-esterase, methyl-esterase,poly galacturanase, endo polyglucanase, and exo polyglucanase. 7.(canceled)
 8. The process according to claim 1, wherein prior to theextraction, the coffee pulp is subjected to a washing process, whereinthe washing process comprises mixing the coffee pulp with a solvent andsubsequently removing at least part of the solvent, wherein the watercontent of the solvent is ≦80 wt. %, wherein at least 50 wt. %,especially at least 80 wt. %, of the solvent consists of one or moreliquids having a polarity lower than water, and wherein prior to theextraction the pulp is subjected to a preservation process, wherein thepreservation process comprises one or more of (i) adding a preservationagent to the pulp and (ii) drying the pulp.
 9. (canceled)
 10. (canceled)11. The process according to claim 1, wherein the extraction ofpolyphenol functionalized coffee pectin extract from the enzymaticallytreated pectin material comprises mixing at least part of theenzymatically treated material with an extraction liquid andsubsequently removing at least part of the polyphenol functionalizedcoffee pectin, wherein the extraction liquid has a pH in the range of4-6.
 12. A polyphenol functionalized pectin.
 13. A polyphenolfunctionalized coffee pectin.
 14. A polyphenol functionalized coffeepectin obtainable by the process according to claim
 1. 15. Thepolyphenol functionalized coffee pectin according to claim 13, having amolar ratio of phenolic groups to the sum of arabinose plus galactoseunits between 20% to 60%, and having a molecular weight >90,000 Da, thepolyphenol functionalized coffee pectin further having a degree ofmethylation (DM) of >75% and a degree of acetylation (DAc) >75%, whereinthe protein content is in the range of 5-18 wt. % and wherein thepolyphenol content is in the range of 0.06-0.18 wt. %.
 16. (canceled)17. The polyphenol functionalized coffee pectin according to claim 13,having a molar weight in the range of 90,000-120,000 Da, having a totalsugar content of rhamnose, arabinose, xylose, mannose, galactose,glucose, galacturonic acid, relative to the total sugar content, in therange of 70-95 wt. %, with a total galacturonic acid content, relativeto the total sugar content, in the range of 55 to 80 wt. %, and having atotal glucose content, relative to the total sugar content, in the rangeof 3-15 wt. %.
 18. A process for producing a pectin based productcomprising using the polyphenol functionalized coffee pectin extractobtainable by the process as defined in claim 13 and processing thepolyphenol functionalized coffee pectin extract together with one ormore other components into the pectin based product.
 19. The processaccording to claim 18, wherein the pectin based product comprises a foodproduct, a pharmaceutical product or a neutraceutical product.